US6534477B2 - Production and use of modified cystatins - Google Patents

Production and use of modified cystatins Download PDF

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US6534477B2
US6534477B2 US09/775,932 US77593201A US6534477B2 US 6534477 B2 US6534477 B2 US 6534477B2 US 77593201 A US77593201 A US 77593201A US 6534477 B2 US6534477 B2 US 6534477B2
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ser
gln
lys
ala
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Shuryo Nakai
Masahiro Ogawa
Soichiro Nakamura
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University of British Columbia
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin

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  • the present invention relates to protease inhibitors, specifically to cystatins, that have been modified by glycosylation in order to enhance stability and activity; to methods of making such modified protease inhibitors and to methods of using such modified protease inhibitors to inhibit proteolysis of a protein substrate.
  • Proteases are enzymes that degrade proteins. Proteases are classified by the substrate upon which they act and include serine proteases, cysteine proteases, aspartate proteases and metalloproteinases. Serine and cysteine proteases are widespread and are found in diverse organisms including eukaryotic and prokaryotic animals and plants. Cysteine proteases are generally well characterized enzymes having a known primary structure composed of alpha helices and beta pleated sheets (8).
  • Proteases mediate many processes that are harmful to man, either by producing pathology or by causing economic loss, for instance by degrading foods.
  • Protease-mediated pathology is known to be caused by a wide variety of organisms including bacteria, such as staphylococci and streptococci, fungi, arthropods, nematodes, protozoa such as amoebas, intestinal flagellates, haemoflagelates, such as Leishmania and trypanosomes and helminths.
  • Proteases are known to be important in the pathology of certain viruses (9, 11, 12, 31) including Polio virus, Herpes virus, Corona virus, HIV and Rotavirus. Proteases are also known to play a role in various diseases with no clear etiological agent, such as muscular dystrophy (7) and cancers (1) including breast cancer (2), and amyloid angiopathy, a genetic disease that often leads to fatal cerebral hemorrhages in young adults (10, 13).
  • Proteases are also responsible for the spoilage of economically important foodstuffs, necessitating huge annual expenditures on preventative measures.
  • Botrytis cinera causes widespread disease in over thirty species of commercial crop plants. Molds and fungi are the major destroyers of citrus fruit crops. Foods rich in protein, such as meat and fish, are degraded and made inedible by proteases from Pseudomonads and other bacteria. Foods with a high muscle content may be quickly broken down by endogenous proteases released from tissues upon death. These enzymes degrade myosin, and destroy the texture of the food.
  • surimi is a form of processed minced fish, commonly made from Pacific Whiting ( Merluccius productus ), and is the main ingredient of seafood analogs such as “imitation crab meat”.
  • Surimi is an important source of relatively cheap, high quality, low fat protein important to the diet of many people in the Far East and of increasing economic significance worldwide. Endogenous proteolytic enzymes released during surimi production cause rapid degradation of muscle tissue and lead to poor quality surimi (3, 4, 5, 6, 11, 17, 18, 19). It is thought that the protease released from the fish tissue is a cathepsin, which is a common cysteine protease (3).
  • protease inhibitors have been investigated for their potential role in preventing disease and degradation of foodstuffs (4, 5, 18).
  • Partially refined substances that contain protease inhibitors are commonly used in food processing, for instance, to prevent proteolytic breakdown of fish protein during the production of surimi (4, 17, 18).
  • the most commonly used food-grade protease inhibitors are beef plasma protein (BPP), egg white powder and potato powder (4).
  • BPP beef plasma protein
  • egg white powder egg white powder
  • potato powder (4).
  • Genetic engineering techniques have been used to introduce protease-inhibitor genes from chickens into cereal and grass plants to control protease-producing plant pathogens.
  • a plant protease inhibitor gene, from Cowpea has been recombinantly introduced into tobacco, tomato, cotton and other plants to inhibit destruction by nematode worms (16).
  • Cystatins are cysteine protease inhibitors that are members of Family 2 of the cystatin superfamily, characterized by a single chain of about 115 to 122 amino acids with a molecular weight of about 13000, having two disulfide bonds (7, 8, 10). Cystatins and protease enzymes such as cathepsins form tight (but reversible) enzyme-inhibitor complexes with dissociation constants typically in the nannomolar range (10).
  • cystatins A number of cystatins have been characterized including human cystatins (C, S, SN, SA, D, M and E), mouse cystatin, egg-white cystatin, bovine cystatin, carp cystatin, trout cystatin and salmon cystatin.
  • cystatins protect the body by inhibiting the potentially harmful effects of proteolysis, and may prevent destruction of connective tissue by protease enzymes, for instance, lysosomal proteases, released from dying or damaged cells (16).
  • Cystatins have been investigated for potential medical applications, for instance, to inhibit replication and pathology of Picornaviruses (12), Coronaviruses (9) and Herpes Simplex type 1 virus (15, 16, 30). Cystatins may also play a natural role in prevention of bacterial infection by E. coli , Shigella (13), Leishmania, Schistosoma and Entamobea (10) which appear to use proteases to facilitate tissue invasion.
  • Cystatins are likely the primary protease inhibitors in food-grade protease inhibitor preparations such as beef plasma protein and egg white powder. Since cystatins are themselves proteins, they are prone to denaturation and loss of activity when exposed to unfavorable temperatures or pH. Many food production processes, including surimi production, involve elevated temperatures (17). Presently, in order to maintain cystatin activity, more cystatin must be added after cooling. Adding additional cystatin is both labor-intensive and expensive. Also, when cystatins are used for medical treatment, either as a topical or ingested medication, it is preferable for the cystatin-containing composition to be sterile. A common method of sterilization involves treatment with elevated temperatures. A cystatin that could maintain activity despite exposure to elevated temperatures would thus be useful in food processing and in drug formulation.
  • the present invention provides modified, glycosylated, heat-stable cystatins and methods of making and using these cystatins.
  • the present invention also provides nucleic acid molecules encoding such cystatins.
  • the nucleic acid molecules of the invention have been modified so that when such a nucleic acid molecule is expressed in a eukaryotic cell, certain amino-acid residues of the expressed cystatin protein are glycosylated during post-translational modification of the protein.
  • the resulting mature protein has attached, at specific amino acid residues, sugar molecule chains of varying length.
  • the present invention includes the nucleic acid molecules that encode modified cystatins based on the cystatins from humans (C, S, SN, SA, D, M and E), egg white, cow, carp, trout and salmon.
  • cystatin primary amino acid sequence Various residues in the cystatin primary amino acid sequence have been identified where the introduction glycosylated residues increases heat stability of the expressed protein without severely affecting enzymatic activity.
  • the sites for glycosylation include amino acid residues at positions 35, 36 and 79.
  • the present invention also includes a method of making modified heat-stable cystatins by modifying the nucleic acid molecules that encode cystatins. Such nucleic acid molecules are modified at certain defined sites and expressed in a eukaryotic cells.
  • the present invention also includes a cell that contains at least one nucleic acid molecule encoding at least one modified, glycosylated, heat-stable cystatin.
  • the cells of the invention may be of many types, for instance they may be cells from a yeast, a mammal, an insect, or a plant.
  • the invention also includes methods of inhibiting proteolysis of a protein substrate by contacting the protein substrate with a modified heat-stable cystatin having at least one engineered glycosylation site. Such a method may be applied, for example, to food processing, such as the production of surimi.
  • the invention also includes a method of treating a protease-mediated pathology of an organism, such as a mammal, a fish or a plant by administering to the organism a modified heat-stable cystatin of the invention.
  • a modified heat-stable cystatin of the invention contacts the protease that mediates the pathology, thereby inhibiting proteolysis by the protease and thereby treating the pathology.
  • SEQ ID NO: 1 shows the cDNA sequence and the amino acid sequence of native human cystatin C.
  • SEQ ID NO: 3 shows the cDNA sequence and the amino acid sequence of native human cystatin S.
  • SEQ ID NO: 4 shows the amino acid sequence of native human cystatin S.
  • SEQ ID NO: 5 shows the cDNA sequence and the amino acid sequence of native human cystatin SN.
  • SEQ ID NO: 6 shows the amino acid sequence of native human cystatin SN.
  • SEQ ID NO: 7 shows the cDNA sequence and the amino acid sequence of native human cystatin SA.
  • SEQ ID NO: 8 shows the amino acid sequence of native human cystatin SA.
  • SEQ ID NO: 9 shows the cDNA sequence and the amino acid sequence of native human cystatin D.
  • SEQ ID NO: 10 shows the amino acid sequence of native human cystatin D.
  • SEQ ID NO: 11 shows the cDNA sequence and the amino acid sequence of native human cystatin M.
  • SEQ ID NO: 12 shows the amino acid sequence of native human cystatin M.
  • SEQ ID NO: 13 shows the cDNA sequence and the amino acid sequence of native human cystatin E.
  • SEQ ID NO: 14 shows the amino acid sequence of native human cystatin E.
  • SEQ ID NO: 15 shows the cDNA sequence and the amino acid sequence of native egg white cystatin.
  • SEQ ID NO: 16 shows the amino acid sequence of native egg white cystatin.
  • SEQ ID NO: 17 shows the cDNA sequence and the amino acid sequence of native carp cystatin.
  • SEQ ID NO: 19 shows the cDNA sequence and the amino acid sequence of native salmon cystatin.
  • SEQ ID NO: 20 shows the amino acid sequence of native salmon cystatin.
  • SEQ ID NO: 21 shows the cDNA sequence and the amino acid sequence of native trout cystatin.
  • SEQ ID NO: 24 shows the amino acid sequence of native bovine cystatin.
  • SEQ ID NO: 25 shows the first of four oligonucleotides used to create a nucleotide coding for modified human cystatin C.
  • SEQ ID NO: 26 shows the second of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C.
  • SEQ ID NO: 27 shows the third of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C.
  • SEQ ID NO: 28 shows the fourth of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C.
  • SEQ ID NO: 29 shows a forward primer used in site-directed mutagenesis to introduce a glycosylation site at residue 35 of a modified human cystatin C.
  • SEQ ID NO: 30 is a reverse primer used in site-directed mutagenesis to introduce a glycosylation site at residue 35 of a modified human cystatin C.
  • SEQ ID NO: 32 is the reverse primer used in site-directed mutagenesis to introduce a glycosylation site at residue 36 of a modified human cystatin C.
  • FIG. 1 shows the native amino acid sequence of the mature human cystatin C peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
  • FIG. 2 shows the native amino acid sequence of the mature human cystatin S peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
  • FIG. 3 shows the native amino acid sequence of the mature human cystatin SN peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
  • FIG. 4 shows the native amino acid sequence of the mature human cystatin SA peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
  • FIG. 5 shows the native amino acid sequence of the mature human cystatin D peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
  • FIG. 8 shows the native amino acid sequence of the mature Egg White cystatin peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
  • FIG. 12 shows the native amino acid sequence of the mature chum salmon cystatin without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
  • Heat stability refers to the ability of a protein to function at high temperatures. Proteins typically lose activity as the temperature is raised above their normal in vivo operating range, and generally become denatured as the temperature increases further. A modified protein may be more heat stable than the unmodified form of the protein, meaning that the modified form of the protein retains greater activity at higher temperatures than the unmodified form of the protein.
  • a feature has been introduced means that a non-wild-type feature has been intentionally, artificially added, for instance, a glycosylation site may be said to be introduced into a protein if it has been intentionally, artificially added to the protein, also a nucleotide may be said to be introduced into a cell or other nucleotide sequence when it has been intentionally, artificially added into a cell or sequence, likewise, a deletion may be introduced into a DNA sequence. Likewise, amino acid substitutions, additions and deletions may be introduced into a protein.
  • a glycosylation site is a place on a molecule at which sugars may be added.
  • the addition of sugars (glycosylation) may occur at introduced sites in modified proteins such as at amino acid residue 37 of the modified human cystatin C.
  • X the native amino acid that has been removed
  • # the position of that amino acid in the protein
  • Z the substituted amino acid that has been inserted in place of X.
  • Ala (37) Ser denotes that Alanine at position 37 has been removed and replaced with Serine; likewise, for one-letter code, A (31) N denotes that Alanine at position 31 has been removed and replaced with Asparagine.
  • the numbering system of cystatin proteins in this text reflects that shown in the accompanying figures. While alternative numbering systems are possible, the nomenclature use herein is intended to identify a particular amino acid residue, rather than any residue that happens to be at a given distance from an arbitrary point on an amino acid sequence.
  • An organism refers to any organism of any kingdom, phylum, class, order, family, genus or species.
  • a cancer refers to any neoplastic transformation or any tissue that has undergone neoplasia and includes solid, non-solid, benign and malignant transformed tissue of both plants and animals.
  • a protease-mediated pathology is any pathology that is caused in part or in whole by the action of a protease, for instance, the proteolytic invasion of human tissue by a cancer cell, the proteolytic destruction of human tissue by a bacterium or a protozoan (e.g., Pseudomonas or Leishmania) and the tissue destruction of a plant by a fungus (e.g., Phytopthora infestans ) are all examples of protease-mediated pathologies.
  • a protease for instance, the proteolytic invasion of human tissue by a cancer cell, the proteolytic destruction of human tissue by a bacterium or a protozoan (e.g., Pseudomonas or Leishmania) and the tissue destruction of a plant by a fungus (e.g., Phytopthora infestans ) are all examples of protease-mediated pathologies.
  • a polynucleotide (or a gene or genome) is recombinant means that it has been altered by the addition at some site of non-native nucleic acids or nucleotides, i.e., nucleotides that are not normally found in the particular polynucleotide, or that are not normally found at that site.
  • a human cystatin gene that has been altered by nucleotide substitution is said to be recombinant.
  • a recombinant protein is a protein that is the product of a recombinant polynucleotide and that contains non-native amino acid residues.
  • Nucleic acid probes and primers may readily be prepared based on the nucleic acid sequences provided by this invention.
  • a probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are well known in the field of molecular biology.
  • Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length, which are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a DNA polymerase enzyme.
  • Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
  • Probes and primers as used in the present invention preferably comprise at least 15 nucleotides of the nucleic acid sequences that encode a modified cystatin protein. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise 20, 30 or 40 consecutive nucleotides of the disclosed nucleic acid sequences. Methods for preparing and using probes and primers are described in a number of reference works, for example Sambrook et al.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • purified peptide does not require absolute purity, rather, it is intended as a relative term.
  • a purified cystatin preparation is one in which cystatin is enriched compared to the cystatin in its natural environment, i.e., within a cell.
  • a purified preparation of a cystatin is prepared such that the cystatin represents at least 20% of the portion of the protein content of the preparation. Preparations comprising at least 50%, 75% or at least 90% cystatin (expressed as a percentage of total protein) may be desirable for certain applications.
  • a series of anti-parallel and complementary nucleotides may be synthesized and annealed together to form synthetic double-stranded nucleotide fragment. These fragments may then be ligated together to produce a contiguous double-stranded nucleotide that codes for a particular cystatin having one or more engineered glycosylation sites.
  • the synthetic nucleotides may be amplified in vitro using the Polymerase Chain Reaction (PCR) (27) so that many copies of the nucleotide are available for cloning into a prokaryotic cloning vector (26) or into an expression vector, as explained below.
  • PCR Polymerase Chain Reaction
  • Additional glycosylation sites may be introduced using site-directed mutagenesis to add, delete or substitute particular amino acid residues.
  • site-directed mutagenesis Various standard techniques are known to carry out site-directed mutagenesis (26, chapter 15), and commercial kits are also available such as the QUICKCHANGETM mutagenesis site-directed mutagenesis kit (STRATAGENETM, CA). Nucleotides may be modified or synthesized so as to include restriction enzyme sites that can be used for cloning.
  • the modified nucleotide may be cloned into a standard prokaryotic cloning vector, for example pBR322, pUC18 or pUC19 (26, chapter 1).
  • a standard prokaryotic cloning vector for example pBR322, pUC18 or pUC19 (26, chapter 1).
  • the sequence of the cloned nucleotide may be checked by sequencing using standard methods (26, chapters 1 and 13).
  • the cloned expression vector may then be transformed into a particular cell type and the nucleotide expressed.
  • Many different types of cell may be used to express the modified nucleic acid molecules. Examples of such cells include cells of yeasts, fungi, insects and humans and plants, including transformed and non-transformed cells.
  • common mammalian cells that could be used for the invention include human HeLa cells, SW-527 human cells (ATCC deposit #7940), WISH cells (ATCC deposit #CCL-25), Daudi cells (ATCC deposit #CCL-213), Mandin-Darby bovine kidney cells (ATCC deposit #CCL-22) and Chinese Hamster ovary cells (ATCC deposit #CRL-2092).
  • Reptile cells that may be used include those from Russell's Viper (ATCC deposit #CCL-140). Plant cells that could be used include Chlamydomonas cells (ATCC deposit #30485), Arabidopsis cells (ATCC deposit #54069) and tomato plant cells (ATCC deposit #54003). Many of these cell types are commonly used and are available from the ATCC as well as from commercial suppliers such as PHARMACIATM and INVITROGENTM.
  • Expressed protein may be accumulated within a cell or may be secreted from the cell. Such expressed protein may then be collected and purified. This protein may then be characterized for activity and heat stability and may be used to practice the methods of the invention.
  • the amino acid sequences of the cystatins are shown in their mature form without a signal peptide.
  • the signal peptide is cleaved off during post-translational modification to produce the mature peptide.
  • the invention may be equally practiced with cystatin peptides which retain the signal peptide.
  • the present invention includes a general method of inhibiting proteolysis of a substrate by providing the modified cystatin and by contacting this cystatin with the substrate.
  • a substrate may be any substrate containing protein.
  • a modified cystatin may be used in food processing applications, in agricultural applications and in human and non-human medical applications.
  • a protein substrate such as minced Pacific Whiting (as used for surimi production) may be treated with a modified cystatin of the invention to inhibit proteolysis caused by the release of endogenous protease from the fish tissue.
  • Suggested concentrations for the application of modified cystatin are, for instance, for surimi processing, from about 1 ⁇ g/g to 100 ⁇ g/g of surimi.
  • the present invention also includes methods of using a modified cystatin for the inhibition of proteases for therapeutic purposes.
  • the cystatins of the invention may be used to inhibit tissue destruction and invasion by pathogens such as staphylococci and streptococci.
  • Streptococcus is the etiological agent of the common skin disease impetigo, and certain particularly rapacious “flesh eating” strains of Staphylococcus and Streptococcus have recently received much media attention, particularly because of their multiple antibiotic resistance.
  • cystatins of the invention in preventing bacterial invasion of host-tissues can be measured, for example, by the method of Betts and Finlay (32). This method can be used to determine tissue invasion of green monkey kidney cells (ATCC deposit #CCL-70) by bacteria such as E. coli and Salmonella typhimurium.
  • the modified cystatin may be administered topically or systemically.
  • the cystatin may be formulated with a carrier or pharmacologically acceptable excipient.
  • the cystatin may be mixed with a carrier such as a petroleum-based or lanolin-based oil to form a gel, and administered topically at the site of infection, thereby contacting the modified cystatin with the protease and preventing tissue destruction.
  • the cystatins of the present invention may also be administered systemically, either orally, intravenously, sub-cutaneously, transdermally or by other methods.
  • the cystatin of the present invention may be formulated into a tablet or solution form and administered orally.
  • Formulation of drugs into pills and tablets is well known in the art.
  • Systemic administration may be used to treat infections that cannot be treated topically such as cancer and systemic viral infections of humans and animals.
  • concentrations of modified cystatin in a cream or ointment may be from about 1 ng to 100 mg per gram total weight of ointment, or may be from about 1 ⁇ g to 1 mg total weight, or may be from about 10 ⁇ g to 100 ⁇ g total weight.
  • the amount of modified cystatin per Kg mass of a patient may be from about 1 ng to 100 mg, or may be from about 1 ⁇ g to 1 mg, or may be from about 10 ⁇ g to 100 ⁇ g.
  • the cystatins of the present invention may be used to inhibit proteolysis caused by pathogens of crop plants and fruits.
  • the modified cystatins may be applied to the surface of fruit or crops to inhibit proteases produced by plant pathogens such as fungi, for instance, Botrytis.
  • the modified cystatin may, for instance, be sprayed or painted onto crops, either in a pure form or diluted in a carrier liquid such as water.
  • the cystatins of the invention show superior heat resistance, maintaining a high degree of activity after exposure to elevated temperatures. These heat resistant cystatins may be particularly useful where sterility is required, such as in medical applications where it is generally desirable for medicines to be uncontaminated with biological organisms.
  • the cystatins of the present invention may be formulated into topical ointments or pills which may then be packaged and sterilized and administrated therapeutically.
  • food processing, such as surimi production involves elevated temperatures.
  • the cystatins of the invention are useful in such processes due to their enhanced resistance.
  • FIGS. 2-12 The same is so for all the other cystatins, FIGS. 2-12.
  • the amino acid sequences for native and modified human cystatin SN are shown in FIG. 3 . Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention.
  • the cDNA sequence for native human cystatin SN is shown in SEQ ID NO: 5.
  • the human cystatin SN protein may be modified to introduce one or more glycosylation sites by the introduction of one or more of the following amino acid substitutions: Ala (31) Asn, Ala (38) Ser, Ala (38) Thr, Lys (37) Asn, Lys (81) Asn, Asp (82) Ser, Asp (82) Thr.
  • the amino acid sequences for native and modified human cystatin SA are shown in FIG. 4 . Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention.
  • the cDNA sequence for native human cystatin SA is shown in SEQ ID NO: 7.
  • the human cystatin SA protein may be modified by the introduction of the following amino acid substitutions: Val (31) Asn, Ala (38) Ser, Ala (38) Thr, Lys (37) Asn, Asp (82) Ser, Asp (82) Thr, Leu (81) Asn.
  • the amino acid sequences for native and modified human cystatin D are shown in FIG. 5 . Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention.
  • the cDNA sequence for native human cystatin D is shown in SEQ ID NO: 9.
  • the human cystatin D protein may be modified by the introduction of the following amino acid substitutions: Ala (31) Asn, Val (38) Ser, Val (38) Thr, Asp (42) Ser, Asp (42) Thr, Asp (83) Ser, Asp (83) Thr, Pro (86) Ser, Pro (86) Thr, Gln (90) Ser, Gln (90) Thr, Tyr (44) Asn.
  • the human cystatin M protein may be modified by the introduction of the following amino acid substitutions: Val (35) Asn, Met (40) Asn, Gly (41) Ser, Gly (41) Thr, Ser (42) Asn, Ile (45) Ser, Ile (45) Thr, Arg (78) Asn, Arg (81) Asn, Asp (88) Asn, Leu (89) Asn.
  • the amino acid sequences for native and modified human cystatin E are shown in FIG. 7 . Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention.
  • the cDNA sequence for native human cystatin E is shown in SEQ ID NO: 13.
  • the human cystatin E protein may be modified by the introduction of the following amino acid substitutions: Val (28) Asn, Met (33) Asn, Gly (34) Ser, Gly (34) Thr, Ser (35) Asn, Ile (38) Ser, Ile (38) Thr, Asp (81) Asn, Leu (82) Asn.
  • the amino acid sequences for native and modified egg white cystatin are shown in FIG. 8 .
  • Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein.
  • One or more such amino acid substitutions may be present in the protein of the present invention.
  • the cDNA sequence for native egg white cystatin is shown in SEQ ID NO: 15.
  • the egg white cystatin protein may be modified by the introduction of the following amino acid substitutions: Ala (35) Ser, Ala (35) Thr, Arg (34) Asn, Lys (39) Ser, Lys (39) Thr, Lys (39) Asn, Tyr (40) Asn, Leu (78) Asn, Lys (91) Asn, Tyr (92) Asn.
  • the amino acid sequences for native and modified bovine cystatin are shown in FIG. 9 . Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention.
  • the cDNA sequence for native bovine cystatin is shown in SEQ ID NO: 23.
  • the bovine cystatin protein may be modified by the introduction of the following amino acid substitutions: Ala (29) Asn, Arg (36) Ser, Arg (36) Thr, Lys (35) Asn, Ala (40) Ser, Ala (40) Thr, Tyr (41) Asn, Leu (79) Asn, Asp (80) Ser, Asp (80) Thr, Pro (88) Ser, Pro (88) Thr.
  • the amino acid sequences for native and modified carp cystatin are shown in FIG. 10 .
  • Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein.
  • One or more such amino acid substitutions may be present in the protein of the present invention.
  • the cDNA sequence for native carp cystatin is shown in SEQ ID NO: 17.
  • the trout cystatin protein was modified by the introduction of the following amino acid substitutions: Lys (29) Asn, Lys (30) Ser, Lys (30) Thr, Met (34) Thr, Met (34) Ser, Lys (39) Asn, Lys (88) Asn.
  • the amino acid sequences for native and modified chum salmon cystatin are shown in FIG. 12 . Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention.
  • the CDNA sequence for native chum salmon cystatin is shown in SEQ ID NO: 19.
  • Cloning was done using the pUC19 cloning vector and E. coli using standard gene cloning techniques (26).
  • the yeast strain Pichia pastons KM71 was used to express mammalian genes and constructs.
  • T4 DNA ligase, restriction enzymes, the 7-DEAZA sequencing kit and blunting kit were all purchased from TAKARA SHUZOTM of Kyoto, Japan.
  • the oligonucleotide in vitro mutagenesis system (version 2) was purchased from AMERSHAMTM International.
  • CM TOYOPEARLTM 650M resin was purchased from TOSOHTM of Tokyo.
  • Concanavalin A-sepharose and a-methylmannoside were purchased from PHARMACIATM and from WAKOTM of Tokyo, respectively.
  • Sephadex-G50 was purchased from PHARMACIATM.
  • M13mp19 was used as a vector for CDNA construction.
  • the modified nucleotide sequences were made in two steps.
  • a synthetic double-stranded DNA was constructed that codes for human cystatin C, modified to have a glycosylation site at residue number 79.
  • This DNA was made by chemically synthesizing four oligonucleotides using an automated oligonucleotide synthesizer (SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28).
  • SEQ ID NO: 25 SEQ ID NO: 26
  • SEQ ID NO: 27 and SEQ ID NO: 28 an automated oligonucleotide synthesizer
  • the oligonucleotides were chosen on the basis of the known nucleotide sequence of the native human cystatin C gene and the known codon usage of P. pastoris (Table 1).
  • DNA was sequenced using the Sanger method (26, chapter 13). Polypeptides were sequenced using the Edman degradation method in a gas-phase protein automated sequencer (SHIMADZUTM model PSQ2).
  • the complementary pairs of the four oligonucleotides were annealed together and the resulting double-stranded fragments were ligated using T4 ligase.
  • the resulting synthetic open reading frame contained an XhoI site at the 5′ end and an Xba site at the 3′ end that were used for cloning.
  • the gene product was ligated into pUC19 (26, chapter 1) and sequenced in both directions to check that the sequence was as predicted.
  • N-glycosylation site was introduced at either residue 35 or 36 using the QUICKCHANGETM site-directed mutagenesis kit (STRATAGENETM, CA) according to manufacturers instructions and using the forward and reverse primers shown in SEQ ID NOs: 29-32.
  • QUICKCHANGETM site-directed mutagenesis kit
  • the Pichia transformants were incubated in yeast minimal medium (YMM).
  • the Pichia transformants were grown at 30° C. for one day in 5 mL of YMM, and then subcultured at 30° C. for four days in 500 mnl of fresh YMM. 100% methanol was added into the fresh YMM to a final concentration of 0.5% methanol every 24 hours to maintain induction of the cystatin gene.
  • Recombinant modified human cystatin C was secreted in the Pichia culture media.
  • the extracellular proteins were collected using an ultrafiltration system with 10,000 MW cut-off (PELLICONTM cassette filter, MILLIPORETM, Bedford, Mass.).
  • the crude proteins thus recovered were applied to a column of Q-SEPHAROSE FAST FLOWTM (PHARMACIATM, Upsala, Sweden) equilibrated with a linear gradient of 0-0.5 M NaCl in 20 mM Tris-HCI buffer (pH7.5).
  • the fraction including the cystatin was determined by the inhibitory activity against papain as described below.
  • the fraction was applied to a column of sephacryl S-100 HR (PHARMACIATM) equilibrated with 0.15 M NaCl-20 mM Tris-HCI buffer (pH7.5). The fraction which showed the inhibitory activity was collected.
  • Cystatin activity was assayed by measuring papain inhibitory activity using N ⁇ -Benzoyl-DL-Arg-p-Nitroanilide (Bz—Arg—NA) (34).
  • Bz—Arg—NA N ⁇ -Benzoyl-DL-Arg-p-Nitroanilide
  • 0.1 ml of cystatin sample and 0.1 ml of papain solution were pipetted into 0.1 mL of 50 mM Tris-HCI buffer (pH7.5) containing 100 mM Bz—Arg—NA, 2 mM EDTA, and 5 mM cystein. The solution was incubated for 25 min at 37° C.
  • the reaction was stopped with 0.2 ml of 30% acetic acid, and the nitroaniline liberated by enzymatic activity is quantified by measurement of light absorption of the solution at 410nm.
  • the inhibitory activity was expressed as the amount of enzyme (mg) inhibited by 1 mg of inhibitor (U/mg).
  • Heat resistance assays were performed by heating a solution containing a known amount of cystatin to a controlled temperature for a controlled time; cooling the solution; mixing the cooled cystatin solution with a known amount of papain and protein substrate, and measuring turbidity of the mixture.
  • the recombinant cystatins were heated to 95° C. at a rate of 1° C./min for 30° C. in 50 mM sodium phosphate buffer (pH 7.5). Protein concentration was 1 mg/ml.
  • a glycosylation site at residue 36 #of a modified human cystatin C ⁇ 400> SEQUENCE: 31 ggtgagtaca acaacgcctc taacgacatg # # 30 ⁇ 210> SEQ ID NO 32 ⁇ 211> LENGTH: 30 ⁇ 212> TYPE: DNA ⁇ 213>
  • ORGANISM Artificial Sequence ⁇ 220> FEATURE: ⁇ 223> OTHER INFORMATION: Description of Artificial #Sequence: reverse primer used in site-directed mutagen #esis to intro.
  • a glycosylation site at residue 36 #of a modified human cystatin C ⁇ 400> SEQUENCE: 32 catgtcgtta gaggcgttgt tactcacc # # # 30

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Abstract

Cystatins that have been modified by glycosylation in order to enhance stability and activity are disclosed, as are methods of making such cystatins and methods of using such cystatins to inhibit proteolysis.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation of International Application No. PCT/CA99/00717, filed Aug. 5, 1999, which claims the benefit of U.S. Provisional Application No. 60/095,503, filed Aug. 5, 1998, both of which are herein incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to protease inhibitors, specifically to cystatins, that have been modified by glycosylation in order to enhance stability and activity; to methods of making such modified protease inhibitors and to methods of using such modified protease inhibitors to inhibit proteolysis of a protein substrate.
BACKGROUND
Proteases are enzymes that degrade proteins. Proteases are classified by the substrate upon which they act and include serine proteases, cysteine proteases, aspartate proteases and metalloproteinases. Serine and cysteine proteases are widespread and are found in diverse organisms including eukaryotic and prokaryotic animals and plants. Cysteine proteases are generally well characterized enzymes having a known primary structure composed of alpha helices and beta pleated sheets (8).
Proteases mediate many processes that are harmful to man, either by producing pathology or by causing economic loss, for instance by degrading foods. Protease-mediated pathology is known to be caused by a wide variety of organisms including bacteria, such as staphylococci and streptococci, fungi, arthropods, nematodes, protozoa such as amoebas, intestinal flagellates, haemoflagelates, such as Leishmania and trypanosomes and helminths.
Proteases are known to be important in the pathology of certain viruses (9, 11, 12, 31) including Polio virus, Herpes virus, Corona virus, HIV and Rotavirus. Proteases are also known to play a role in various diseases with no clear etiological agent, such as muscular dystrophy (7) and cancers (1) including breast cancer (2), and amyloid angiopathy, a genetic disease that often leads to fatal cerebral hemorrhages in young adults (10, 13).
Proteases are also responsible for the spoilage of economically important foodstuffs, necessitating huge annual expenditures on preventative measures. For instance, the fungus Botrytis cinera causes widespread disease in over thirty species of commercial crop plants. Molds and fungi are the major destroyers of citrus fruit crops. Foods rich in protein, such as meat and fish, are degraded and made inedible by proteases from Pseudomonads and other bacteria. Foods with a high muscle content may be quickly broken down by endogenous proteases released from tissues upon death. These enzymes degrade myosin, and destroy the texture of the food. An important example of such spoilage occurs during the processing of surimi, which is a form of processed minced fish, commonly made from Pacific Whiting (Merluccius productus), and is the main ingredient of seafood analogs such as “imitation crab meat”. Surimi is an important source of relatively cheap, high quality, low fat protein important to the diet of many people in the Far East and of increasing economic significance worldwide. Endogenous proteolytic enzymes released during surimi production cause rapid degradation of muscle tissue and lead to poor quality surimi (3, 4, 5, 6, 11, 17, 18, 19). It is thought that the protease released from the fish tissue is a cathepsin, which is a common cysteine protease (3).
Because of the role of proteases in these various processes, protease inhibitors have been investigated for their potential role in preventing disease and degradation of foodstuffs (4, 5, 18). Partially refined substances that contain protease inhibitors are commonly used in food processing, for instance, to prevent proteolytic breakdown of fish protein during the production of surimi (4, 17, 18). The most commonly used food-grade protease inhibitors are beef plasma protein (BPP), egg white powder and potato powder (4). Genetic engineering techniques have been used to introduce protease-inhibitor genes from chickens into cereal and grass plants to control protease-producing plant pathogens. Likewise, a plant protease inhibitor gene, from Cowpea, has been recombinantly introduced into tobacco, tomato, cotton and other plants to inhibit destruction by nematode worms (16).
Cystatins are cysteine protease inhibitors that are members of Family 2 of the cystatin superfamily, characterized by a single chain of about 115 to 122 amino acids with a molecular weight of about 13000, having two disulfide bonds (7, 8, 10). Cystatins and protease enzymes such as cathepsins form tight (but reversible) enzyme-inhibitor complexes with dissociation constants typically in the nannomolar range (10).
A number of cystatins have been characterized including human cystatins (C, S, SN, SA, D, M and E), mouse cystatin, egg-white cystatin, bovine cystatin, carp cystatin, trout cystatin and salmon cystatin. In their natural state, cystatins protect the body by inhibiting the potentially harmful effects of proteolysis, and may prevent destruction of connective tissue by protease enzymes, for instance, lysosomal proteases, released from dying or damaged cells (16).
Cystatins have been investigated for potential medical applications, for instance, to inhibit replication and pathology of Picornaviruses (12), Coronaviruses (9) and Herpes Simplex type 1 virus (15, 16, 30). Cystatins may also play a natural role in prevention of bacterial infection by E. coli, Shigella (13), Leishmania, Schistosoma and Entamobea (10) which appear to use proteases to facilitate tissue invasion.
Cystatins are likely the primary protease inhibitors in food-grade protease inhibitor preparations such as beef plasma protein and egg white powder. Since cystatins are themselves proteins, they are prone to denaturation and loss of activity when exposed to unfavorable temperatures or pH. Many food production processes, including surimi production, involve elevated temperatures (17). Presently, in order to maintain cystatin activity, more cystatin must be added after cooling. Adding additional cystatin is both labor-intensive and expensive. Also, when cystatins are used for medical treatment, either as a topical or ingested medication, it is preferable for the cystatin-containing composition to be sterile. A common method of sterilization involves treatment with elevated temperatures. A cystatin that could maintain activity despite exposure to elevated temperatures would thus be useful in food processing and in drug formulation.
SUMMARY OF THE DISCLOSURE
The present invention provides modified, glycosylated, heat-stable cystatins and methods of making and using these cystatins. The present invention also provides nucleic acid molecules encoding such cystatins.
The nucleic acid molecules of the invention have been modified so that when such a nucleic acid molecule is expressed in a eukaryotic cell, certain amino-acid residues of the expressed cystatin protein are glycosylated during post-translational modification of the protein. The resulting mature protein has attached, at specific amino acid residues, sugar molecule chains of varying length. The present invention includes the nucleic acid molecules that encode modified cystatins based on the cystatins from humans (C, S, SN, SA, D, M and E), egg white, cow, carp, trout and salmon.
Various residues in the cystatin primary amino acid sequence have been identified where the introduction glycosylated residues increases heat stability of the expressed protein without severely affecting enzymatic activity. In human cystatin C, for instance, the sites for glycosylation include amino acid residues at positions 35, 36 and 79.
The present invention also includes a method of making modified heat-stable cystatins by modifying the nucleic acid molecules that encode cystatins. Such nucleic acid molecules are modified at certain defined sites and expressed in a eukaryotic cells. The present invention also includes a cell that contains at least one nucleic acid molecule encoding at least one modified, glycosylated, heat-stable cystatin. The cells of the invention may be of many types, for instance they may be cells from a yeast, a mammal, an insect, or a plant.
The invention also includes methods of inhibiting proteolysis of a protein substrate by contacting the protein substrate with a modified heat-stable cystatin having at least one engineered glycosylation site. Such a method may be applied, for example, to food processing, such as the production of surimi.
The invention also includes a method of treating a protease-mediated pathology of an organism, such as a mammal, a fish or a plant by administering to the organism a modified heat-stable cystatin of the invention. By such administration, the modified heat-stable cystatin contacts the protease that mediates the pathology, thereby inhibiting proteolysis by the protease and thereby treating the pathology.
SEQUENCE LISTING
SEQ ID NO: 1 shows the cDNA sequence and the amino acid sequence of native human cystatin C.
SEQ ID NO: 2 shows the amino acid sequence of native human cystatin C.
SEQ ID NO: 3 shows the cDNA sequence and the amino acid sequence of native human cystatin S.
SEQ ID NO: 4 shows the amino acid sequence of native human cystatin S.
SEQ ID NO: 5 shows the cDNA sequence and the amino acid sequence of native human cystatin SN.
SEQ ID NO: 6 shows the amino acid sequence of native human cystatin SN.
SEQ ID NO: 7 shows the cDNA sequence and the amino acid sequence of native human cystatin SA.
SEQ ID NO: 8 shows the amino acid sequence of native human cystatin SA.
SEQ ID NO: 9 shows the cDNA sequence and the amino acid sequence of native human cystatin D.
SEQ ID NO: 10 shows the amino acid sequence of native human cystatin D.
SEQ ID NO: 11 shows the cDNA sequence and the amino acid sequence of native human cystatin M.
SEQ ID NO: 12 shows the amino acid sequence of native human cystatin M.
SEQ ID NO: 13 shows the cDNA sequence and the amino acid sequence of native human cystatin E.
SEQ ID NO: 14 shows the amino acid sequence of native human cystatin E.
SEQ ID NO: 15 shows the cDNA sequence and the amino acid sequence of native egg white cystatin.
SEQ ID NO: 16 shows the amino acid sequence of native egg white cystatin.
SEQ ID NO: 17 shows the cDNA sequence and the amino acid sequence of native carp cystatin.
SEQ ID NO: 18 shows the amino acid sequence of native carp cystatin.
SEQ ID NO: 19 shows the cDNA sequence and the amino acid sequence of native salmon cystatin.
SEQ ID NO: 20 shows the amino acid sequence of native salmon cystatin.
SEQ ID NO: 21 shows the cDNA sequence and the amino acid sequence of native trout cystatin.
SEQ ID NO: 22 shows the amino acid sequence of native trout cystatin.
SEQ ID NO: 23 shows the cDNA sequence and the amino acid sequence of native bovine cystatin.
SEQ ID NO: 24 shows the amino acid sequence of native bovine cystatin.
SEQ ID NO: 25 shows the first of four oligonucleotides used to create a nucleotide coding for modified human cystatin C.
SEQ ID NO: 26 shows the second of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C.
SEQ ID NO: 27 shows the third of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C.
SEQ ID NO: 28 shows the fourth of four oligonucleotides used to create a nucleotide coding for synthetic human cystatin C.
SEQ ID NO: 29 shows a forward primer used in site-directed mutagenesis to introduce a glycosylation site at residue 35 of a modified human cystatin C.
SEQ ID NO: 30 is a reverse primer used in site-directed mutagenesis to introduce a glycosylation site at residue 35 of a modified human cystatin C.
SEQ ID NO: 31 is the forward primer used in site-directed mutagenesis to introduce a glycosylation site at residue 36 of a modified human cystatin C.
SEQ ID NO: 32 is the reverse primer used in site-directed mutagenesis to introduce a glycosylation site at residue 36 of a modified human cystatin C.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the native amino acid sequence of the mature human cystatin C peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
FIG. 2 shows the native amino acid sequence of the mature human cystatin S peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
FIG. 3 shows the native amino acid sequence of the mature human cystatin SN peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
FIG. 4 shows the native amino acid sequence of the mature human cystatin SA peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
FIG. 5 shows the native amino acid sequence of the mature human cystatin D peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
FIG. 6 shows the native amino acid sequence of the mature human cystatin M peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
FIG. 7 shows the native amino acid sequence of the mature human cystatin E peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
FIG. 8 shows the native amino acid sequence of the mature Egg White cystatin peptide without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
FIG. 9 shows the native amino acid sequence of the mature bovine cystatin without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
FIG. 10 shows the native amino acid sequence of the mature carp cystatin without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
FIG. 11 shows the native amino acid sequence of the mature trout cystatin without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
FIG. 12 shows the native amino acid sequence of the mature chum salmon cystatin without its signal sequence. Amino acid modifications for introducing glycosylation sites are shown below the native sequence.
DESCRIPTION OF THE INVENTION
A. Definitions
A protein is said to be modified when it has been intentionally, artificially altered from its naturally occurring, wild-type form, e.g., when the primary amino acid sequence of a cystatin protein has been intentionally, artificially altered to add a non-native glycosylation site somewhere in the protein. A DNA sequence is said to be modified when it has been intentionally, artificially altered from its naturally occurring, wild-type form, e.g., when the nucleotide coding for a cystatin protein has been mutated by site-directed mutagenesis to create a non-native nucleotide addition, deletion of substitution. The term engineered may be used synonymously with the term modified.
To say that an organism, or nucleotide sequence has been genetically engineered means that it has been intentionally, artificially genetically altered from its naturally occurring, wild-type genetic form, for instance, a DNA sequence is said to be genetically engineered when its sequence has been intentionally, artificially genetically altered from its wild-type sequence. Any genetically engineered organism or nucleotide sequence has been modified.
Heat stability refers to the ability of a protein to function at high temperatures. Proteins typically lose activity as the temperature is raised above their normal in vivo operating range, and generally become denatured as the temperature increases further. A modified protein may be more heat stable than the unmodified form of the protein, meaning that the modified form of the protein retains greater activity at higher temperatures than the unmodified form of the protein.
To say a feature has been introduced means that a non-wild-type feature has been intentionally, artificially added, for instance, a glycosylation site may be said to be introduced into a protein if it has been intentionally, artificially added to the protein, also a nucleotide may be said to be introduced into a cell or other nucleotide sequence when it has been intentionally, artificially added into a cell or sequence, likewise, a deletion may be introduced into a DNA sequence. Likewise, amino acid substitutions, additions and deletions may be introduced into a protein.
A glycosylation site is a place on a molecule at which sugars may be added. The addition of sugars (glycosylation) may occur at introduced sites in modified proteins such as at amino acid residue 37 of the modified human cystatin C.
A glycosylation site in a protein is denoted by the general formula X (#) Z, where X=the native amino acid that has been removed, and # =the position of that amino acid in the protein, and Z=the substituted amino acid that has been inserted in place of X. For instance, Ala (37) Ser denotes that Alanine at position 37 has been removed and replaced with Serine; likewise, for one-letter code, A (31) N denotes that Alanine at position 31 has been removed and replaced with Asparagine. The numbering system of cystatin proteins in this text reflects that shown in the accompanying figures. While alternative numbering systems are possible, the nomenclature use herein is intended to identify a particular amino acid residue, rather than any residue that happens to be at a given distance from an arbitrary point on an amino acid sequence.
An organism refers to any organism of any kingdom, phylum, class, order, family, genus or species.
A pathology refers to a state that is measurably or detectably at variance with normal, healthy, non-disease-state physiology.
A cancer refers to any neoplastic transformation or any tissue that has undergone neoplasia and includes solid, non-solid, benign and malignant transformed tissue of both plants and animals.
A protease-mediated pathology is any pathology that is caused in part or in whole by the action of a protease, for instance, the proteolytic invasion of human tissue by a cancer cell, the proteolytic destruction of human tissue by a bacterium or a protozoan (e.g., Pseudomonas or Leishmania) and the tissue destruction of a plant by a fungus (e.g., Phytopthora infestans) are all examples of protease-mediated pathologies.
To say that a polynucleotide (or a gene or genome) is recombinant means that it has been altered by the addition at some site of non-native nucleic acids or nucleotides, i.e., nucleotides that are not normally found in the particular polynucleotide, or that are not normally found at that site. For instance, a human cystatin gene that has been altered by nucleotide substitution is said to be recombinant. Likewise, a recombinant protein is a protein that is the product of a recombinant polynucleotide and that contains non-native amino acid residues.
An isolated nucleic acid has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism in which the nucleic acid naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA. The term isolated thus encompasses nucleic acids purified by standard nucleic acid purification methods. The term also embraces nucleic acids prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
Nucleic acid probes and primers may readily be prepared based on the nucleic acid sequences provided by this invention. A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are well known in the field of molecular biology. Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length, which are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art. Probes and primers as used in the present invention preferably comprise at least 15 nucleotides of the nucleic acid sequences that encode a modified cystatin protein. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise 20, 30 or 40 consecutive nucleotides of the disclosed nucleic acid sequences. Methods for preparing and using probes and primers are described in a number of reference works, for example Sambrook et al. (1989) (26); Ausubel et al., (1987) (25); Innis et al., (1990) (27). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).
A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
The term purified peptide does not require absolute purity, rather, it is intended as a relative term. Thus, for example, a purified cystatin preparation is one in which cystatin is enriched compared to the cystatin in its natural environment, i.e., within a cell. A purified preparation of a cystatin is prepared such that the cystatin represents at least 20% of the portion of the protein content of the preparation. Preparations comprising at least 50%, 75% or at least 90% cystatin (expressed as a percentage of total protein) may be desirable for certain applications.
B. General Methods
The present invention utilizes standard laboratory practices for the cloning, manipulation and sequencing of nucleic acids, purification and analysis of proteins and other molecular biological and biochemical techniques, unless otherwise stipulated. Such techniques are explained in detail in standard laboratory manuals such as Sambrook et al., (1989)(25) and Ausubel et al., (1987)(26).
(1) Production, cloning and expression of modified cystatins
A modified nucleotide sequence that codes for a cystatin protein having at least one glycosylation site may be produced by making synthetic sequences using a commercial polynucleotide synthesizer. The synthetic nucleotides are designed on the basis of the known nucleotide sequence of the cystatin gene and the known codon usage for the cell type in which the nucleotides are to be expressed. A suitable target site for the introduction of a glycosylation site is identified based on the primary amino acid sequence of the cystatin to be modified. A nucleotide sequence is designed that will code for a peptide that includes such a site. The synthetic nucleotide sequence may then be synthesized as described herein. A series of anti-parallel and complementary nucleotides may be synthesized and annealed together to form synthetic double-stranded nucleotide fragment. These fragments may then be ligated together to produce a contiguous double-stranded nucleotide that codes for a particular cystatin having one or more engineered glycosylation sites. The synthetic nucleotides may be amplified in vitro using the Polymerase Chain Reaction (PCR) (27) so that many copies of the nucleotide are available for cloning into a prokaryotic cloning vector (26) or into an expression vector, as explained below.
Additional glycosylation sites may be introduced using site-directed mutagenesis to add, delete or substitute particular amino acid residues. Various standard techniques are known to carry out site-directed mutagenesis (26, chapter 15), and commercial kits are also available such as the QUICKCHANGE™ mutagenesis site-directed mutagenesis kit (STRATAGENE™, CA). Nucleotides may be modified or synthesized so as to include restriction enzyme sites that can be used for cloning.
The modified nucleotide may be cloned into a standard prokaryotic cloning vector, for example pBR322, pUC18 or pUC19 (26, chapter 1). The sequence of the cloned nucleotide may be checked by sequencing using standard methods (26, chapters 1 and 13).
Modified nucleotides may be cloned into an expression vector that allows protein production in a particular cell type. Since the proteins of the invention are glycosylated, it is required that the cell type in which the proteins are expressed can readily carry out post-translational modification including glycosylation. Typically, a eukaryotic cell is used that glycosylates peptides at the Asn-X-Ser/Thr motif. Yeast cells are commonly used for such a purpose. Standard cloning techniques may be used (26, chapter 9). Such expression vector/cell systems are well known and commercially available and include vector/cell combinations that carry out post-translational modifications required for the proper expression of glycosylated eukaryotic proteins. Various yeast strains and yeast-derived vectors are commonly used for this type of expression, for instance, Pichia pastoris expression systems that may be used to practice the present invention may be obtained from INVITROGEN™. Such systems include suitable Pichia pastoris strains, vectors, reagents, transformants, sequencing primers and media. Available strains include a GS115 his 4 deficient strain, a KM71 aox1 deficient strain, a GS115 His+ Mut− strain for extracellular expression and a His+ Mut− strain for intracellular expression (33).
Non-yeast eukaryotic vectors may equally be used for expression of the modified nucleotides. Mammalian vector / host cell systems that contain genetic and cellular control elements capable of carrying out transcription, translation and post-translational modification are well known in the art. Examples of such systems are the well known Baculovirus system, the Ecdysone-inducible mammalian expression system that uses regulatory elements from Drosophila melanogaster to allow control of gene expression, and the Sindbis viral expression system that allows high level expression in a variety of mammalian cell lines, which are available from INVITROGEN™.
The cloned expression vector may then be transformed into a particular cell type and the nucleotide expressed. Many different types of cell may be used to express the modified nucleic acid molecules. Examples of such cells include cells of yeasts, fungi, insects and humans and plants, including transformed and non-transformed cells. For instance, common mammalian cells that could be used for the invention include human HeLa cells, SW-527 human cells (ATCC deposit #7940), WISH cells (ATCC deposit #CCL-25), Daudi cells (ATCC deposit #CCL-213), Mandin-Darby bovine kidney cells (ATCC deposit #CCL-22) and Chinese Hamster ovary cells (ATCC deposit #CRL-2092). Common yeast cells include Pichia pastoris (ATCC deposit #201178) and Saccharomyces cerevisiae (ATCC deposit #46024). Insect cells include cells from Drosophila melanogaster (ATCC deposit #CRL-10191), the cotton bollworm (ATCC deposit #CRL-9281) and from Trichoplusia ni egg cell homoflagelates. Fish cells that may be used include those from rainbow trout (ATCC deposit #CLL-55), salmon (ATCC deposit #CRL-1681) and Zebrafish (ATCC deposit #CRL-2147). Amphibian cells that may be used include those of the Bullfrog, Rana catesbelana (ATCC deposit #CLL-41). Reptile cells that may be used include those from Russell's Viper (ATCC deposit #CCL-140). Plant cells that could be used include Chlamydomonas cells (ATCC deposit #30485), Arabidopsis cells (ATCC deposit #54069) and tomato plant cells (ATCC deposit #54003). Many of these cell types are commonly used and are available from the ATCC as well as from commercial suppliers such as PHARMACIA™ and INVITROGEN™.
Expressed protein may be accumulated within a cell or may be secreted from the cell. Such expressed protein may then be collected and purified. This protein may then be characterized for activity and heat stability and may be used to practice the methods of the invention.
The amino acid sequences of the cystatins (FIGS. 1-12) are shown in their mature form without a signal peptide. The signal peptide is cleaved off during post-translational modification to produce the mature peptide. The invention may be equally practiced with cystatin peptides which retain the signal peptide.
(2) Measurement of protease activity and heat resistance
Protease inhibition activity of modified cystatins is assayed by measuring the reduction in activity of the protease papain. A substrate is used that releases nitroaniline into the reaction medium. The amount of nitroaniline released is determined by measuring light absorption by the solution. This assay is described in detail in Example 4, below.
Heat resistance of the modified cystatin is determined by heating a solution containing a known amount of modified cystatin, cooling it, and adding it to a mixture containing a known amount of protease and a known amount of protein. Protease activity is measured by determining turbidity of the mixture after a set time and also by using the nitroaniline assay as described below.
(3) Methods of using modified cystatins
The modified cystatins of the invention may be used to inhibit proteolysis of a protein substrate in generally the same way as non-modified cystatins are used. Such uses include inhibition of proteolysis in food processing (4), therapeutic treatment of viral disease such as those caused by Herpes Simplex (11, 30), picornaviruses (20) and coronavirus (9).
The present invention includes a general method of inhibiting proteolysis of a substrate by providing the modified cystatin and by contacting this cystatin with the substrate. Such a substrate may be any substrate containing protein. Such a modified cystatin may be used in food processing applications, in agricultural applications and in human and non-human medical applications. For instance, a protein substrate such as minced Pacific Whiting (as used for surimi production) may be treated with a modified cystatin of the invention to inhibit proteolysis caused by the release of endogenous protease from the fish tissue. Suggested concentrations for the application of modified cystatin are, for instance, for surimi processing, from about 1 μg/g to 100 μg/g of surimi.
The present invention also includes methods of using a modified cystatin for the inhibition of proteases for therapeutic purposes. For instance, the cystatins of the invention may be used to inhibit tissue destruction and invasion by pathogens such as staphylococci and streptococci. Streptococcus is the etiological agent of the common skin disease impetigo, and certain particularly rapacious “flesh eating” strains of Staphylococcus and Streptococcus have recently received much media attention, particularly because of their multiple antibiotic resistance.
The effectiveness of cystatins of the invention in preventing bacterial invasion of host-tissues can be measured, for example, by the method of Betts and Finlay (32). This method can be used to determine tissue invasion of green monkey kidney cells (ATCC deposit #CCL-70) by bacteria such as E. coli and Salmonella typhimurium.
For medical applications the modified cystatin may be administered topically or systemically. The cystatin may be formulated with a carrier or pharmacologically acceptable excipient. For instance, the cystatin may be mixed with a carrier such as a petroleum-based or lanolin-based oil to form a gel, and administered topically at the site of infection, thereby contacting the modified cystatin with the protease and preventing tissue destruction.
The cystatins of the present invention may also be administered systemically, either orally, intravenously, sub-cutaneously, transdermally or by other methods. For instance, the cystatin of the present invention may be formulated into a tablet or solution form and administered orally. Formulation of drugs into pills and tablets is well known in the art. Systemic administration may be used to treat infections that cannot be treated topically such as cancer and systemic viral infections of humans and animals.
For topical medical treatment, concentrations of modified cystatin in a cream or ointment may be from about 1 ng to 100 mg per gram total weight of ointment, or may be from about 1 μg to 1 mg total weight, or may be from about 10 μg to 100 μg total weight. For ingested medications, the amount of modified cystatin per Kg mass of a patient may be from about 1 ng to 100 mg, or may be from about 1 μg to 1 mg, or may be from about 10 μg to 100 μg.
The cystatins of the present invention may be used to inhibit proteolysis caused by pathogens of crop plants and fruits. For instance, the modified cystatins may be applied to the surface of fruit or crops to inhibit proteases produced by plant pathogens such as fungi, for instance, Botrytis. The modified cystatin may, for instance, be sprayed or painted onto crops, either in a pure form or diluted in a carrier liquid such as water.
The cystatins of the invention show superior heat resistance, maintaining a high degree of activity after exposure to elevated temperatures. These heat resistant cystatins may be particularly useful where sterility is required, such as in medical applications where it is generally desirable for medicines to be uncontaminated with biological organisms. For instance, the cystatins of the present invention may be formulated into topical ointments or pills which may then be packaged and sterilized and administrated therapeutically. Also, food processing, such as surimi production, involves elevated temperatures. The cystatins of the invention are useful in such processes due to their enhanced resistance.
C. Production of Modified Human Cystatin C
Glycosylation of one or more amino acid residues at specific sites in the human cystatin C peptide increases heat stability of the cystatin without substantially inhibiting functionality. Glycosylation at other, inappropriate sites, may drastically decrease or destroy the protein's inhibitory function. The invention identifies three amino acid residues of human cystatin C that may be modified to introduce a glycosylation site; one or more of these sites may be modified to produce a cystatin protein having superior properties, such as enhanced heat stability.
The amino acid sequences for native and modified human cystatin C are shown in FIG. 1. Amino acid substitutions to produce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more of these amino acid substitutions may be present in a modified cystatin protein of the present invention. The cDNA sequence for native human cystatin C is shown in SEQ ID NO: 1.
Human cystatin C protein may be modified to introduce glycosylation sites (Asn-X-Ser/Thr) at amino acid residues 35, 36 or 79 by the introduction of amino acid substitutions at positions 37, 36 or 81, respectively. One or more of the following specific substitutions may be made: Ala (37) Ser, Ala (37) Thr, Lys (36) Asn, Asp (81) Ser, Asp (81) Thr.
It should be noted that the peptide of cystatin C in FIG. 1 is shown in its mature form without a signal peptide. The signal peptide is cleaved off during post-translational modification to produce the mature peptide. The invention may be equally practiced with cystatin peptides which retain the signal peptide.
The same is so for all the other cystatins, FIGS. 2-12.
D. Production of Modified Human Cystatin S
The amino acid sequences for native and modified human cystatin S are shown in FIG. 2. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin S is shown in SEQ ID NO: 3.
The human cystatin S protein may be modified by the introduction of the following amino acid substitutions: Ala (31) Asn, Lys (37) Asn, Ala (38) Ser, Ala (38) Thr, Leu (81) Asn, Asp (82) Thr, and Asp (82) Ser.
E. Production of Modified Human Cystatin SN
The amino acid sequences for native and modified human cystatin SN are shown in FIG. 3. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin SN is shown in SEQ ID NO: 5.
The human cystatin SN protein may be modified to introduce one or more glycosylation sites by the introduction of one or more of the following amino acid substitutions: Ala (31) Asn, Ala (38) Ser, Ala (38) Thr, Lys (37) Asn, Lys (81) Asn, Asp (82) Ser, Asp (82) Thr.
F. Production of Modified Human Cystatin SA
The amino acid sequences for native and modified human cystatin SA are shown in FIG. 4. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin SA is shown in SEQ ID NO: 7.
The human cystatin SA protein may be modified by the introduction of the following amino acid substitutions: Val (31) Asn, Ala (38) Ser, Ala (38) Thr, Lys (37) Asn, Asp (82) Ser, Asp (82) Thr, Leu (81) Asn.
G. Production of Modified Human Cystatin D
The amino acid sequences for native and modified human cystatin D are shown in FIG. 5. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin D is shown in SEQ ID NO: 9.
The human cystatin D protein may be modified by the introduction of the following amino acid substitutions: Ala (31) Asn, Val (38) Ser, Val (38) Thr, Asp (42) Ser, Asp (42) Thr, Asp (83) Ser, Asp (83) Thr, Pro (86) Ser, Pro (86) Thr, Gln (90) Ser, Gln (90) Thr, Tyr (44) Asn.
H. Production of Modified Human Cystatin M
The amino acid sequences for native and modified human cystatin M are shown in FIG. 6. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin M is shown in SEQ ID NO: 11.
The human cystatin M protein may be modified by the introduction of the following amino acid substitutions: Val (35) Asn, Met (40) Asn, Gly (41) Ser, Gly (41) Thr, Ser (42) Asn, Ile (45) Ser, Ile (45) Thr, Arg (78) Asn, Arg (81) Asn, Asp (88) Asn, Leu (89) Asn.
I. Production of Modified Human Cystatin E
The amino acid sequences for native and modified human cystatin E are shown in FIG. 7. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native human cystatin E is shown in SEQ ID NO: 13.
The human cystatin E protein may be modified by the introduction of the following amino acid substitutions: Val (28) Asn, Met (33) Asn, Gly (34) Ser, Gly (34) Thr, Ser (35) Asn, Ile (38) Ser, Ile (38) Thr, Asp (81) Asn, Leu (82) Asn.
J. Production of Modified Egg White Cystatin
The amino acid sequences for native and modified egg white cystatin are shown in FIG. 8. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native egg white cystatin is shown in SEQ ID NO: 15.
The egg white cystatin protein may be modified by the introduction of the following amino acid substitutions: Ala (35) Ser, Ala (35) Thr, Arg (34) Asn, Lys (39) Ser, Lys (39) Thr, Lys (39) Asn, Tyr (40) Asn, Leu (78) Asn, Lys (91) Asn, Tyr (92) Asn.
K. Production of Modified Bovine Cystatin
The amino acid sequences for native and modified bovine cystatin are shown in FIG. 9. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native bovine cystatin is shown in SEQ ID NO: 23.
The bovine cystatin protein may be modified by the introduction of the following amino acid substitutions: Ala (29) Asn, Arg (36) Ser, Arg (36) Thr, Lys (35) Asn, Ala (40) Ser, Ala (40) Thr, Tyr (41) Asn, Leu (79) Asn, Asp (80) Ser, Asp (80) Thr, Pro (88) Ser, Pro (88) Thr.
L. Production of Modified Carp Cystatin
The amino acid sequences for native and modified carp cystatin are shown in FIG. 10. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native carp cystatin is shown in SEQ ID NO: 17.
The carp cystatin protein may be modified by the introduction of the following amino acid substitutions: Gln (31) Ser, Gln (31) Thr, Gly (30) Asn, Ala (35) Ser, Ala (35) Thr, Lys (39) Asn, Lys (91) Asn.
M. Production of Modified Trout Cystatin
The amino acid sequences for native and modified trout cystatin are shown in FIG. 11. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The cDNA sequence for native trout cystatin is shown in SEQ ID NO: 21.
The trout cystatin protein was modified by the introduction of the following amino acid substitutions: Lys (29) Asn, Lys (30) Ser, Lys (30) Thr, Met (34) Thr, Met (34) Ser, Lys (39) Asn, Lys (88) Asn.
N. Production of Modified Chum Salmon Cystatin
The amino acid sequences for native and modified chum salmon cystatin are shown in FIG. 12. Amino acid substitutions to introduce glycosylation sites are shown directly beneath the amino acids of the native protein. One or more such amino acid substitutions may be present in the protein of the present invention. The CDNA sequence for native chum salmon cystatin is shown in SEQ ID NO: 19.
The chum salmon cystatin protein may be modified by the introduction of the following amino acid substitutions: Lys (29) Asn, Lys (30) Ser, Lys (30) Thr, Met (34) Ser, Met (34) Thr, Lys (88) Asn.
EXAMPLE 1 Synthesis of Nucleotide Sequences that Code for Modified Human Cystatin C
Cloning was done using the pUC19 cloning vector and E. coli using standard gene cloning techniques (26). The yeast strain Pichia pastons KM71 was used to express mammalian genes and constructs. T4 DNA ligase, restriction enzymes, the 7-DEAZA sequencing kit and blunting kit were all purchased from TAKARA SHUZO™ of Kyoto, Japan. The oligonucleotide in vitro mutagenesis system (version 2) was purchased from AMERSHAM™ International. CM TOYOPEARL™ 650M resin was purchased from TOSOH™ of Tokyo. Concanavalin A-sepharose and a-methylmannoside were purchased from PHARMACIA™ and from WAKO™ of Tokyo, respectively. Sephadex-G50 was purchased from PHARMACIA™. M13mp19 was used as a vector for CDNA construction.
The modified nucleotide sequences were made in two steps.
First, a synthetic double-stranded DNA was constructed that codes for human cystatin C, modified to have a glycosylation site at residue number 79. This DNA was made by chemically synthesizing four oligonucleotides using an automated oligonucleotide synthesizer (SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28). The oligonucleotides were chosen on the basis of the known nucleotide sequence of the native human cystatin C gene and the known codon usage of P. pastoris (Table 1).
DNA was sequenced using the Sanger method (26, chapter 13). Polypeptides were sequenced using the Edman degradation method in a gas-phase protein automated sequencer (SHIMADZU™ model PSQ2).
TABLE I
Amino acid Codon usage
Glycine GGT or GGA
Glutamic acid GAG of GAA
Aspartic acid GAC or GAT
Valine GTT or GTC
Alanine GCT or GCC
Arginine AGA or CGT
Serine TCT or TCC
Lysine AAG
Asparagine AAC
Methionine ATG
Isoleucine ATT or ATC
Threonine ACT or ACC
Tryptophan TGG
Cysteine TGT
Tyrosine TAC
Leucine TTG or CTG
Phenylalanine TTC
Glutamine CAA or CAG
Histidine CAC or CAT
Proline CCA or CCT
The complementary pairs of the four oligonucleotides were annealed together and the resulting double-stranded fragments were ligated using T4 ligase. The resulting synthetic open reading frame contained an XhoI site at the 5′ end and an Xba site at the 3′ end that were used for cloning. The gene product was ligated into pUC19 (26, chapter 1) and sequenced in both directions to check that the sequence was as predicted.
Second, an N-glycosylation site was introduced at either residue 35 or 36 using the QUICKCHANGE™ site-directed mutagenesis kit (STRATAGENE™, CA) according to manufacturers instructions and using the forward and reverse primers shown in SEQ ID NOs: 29-32.
EXAMPLE 2 Transformation and Clone Selection for Production of Modified Human Cystatin C
The yeast expression plasmids pYG-100 (20) and pPICZ α-C containing Saccharomyces cerevissiae α factor secretion signal and alcohol oxidase (AOX1) gene promoter were used to express the proteins of the invention. P. pastoris strain KM71 was transformed using the Pichia EASYCOM™ transformation system (INVITROGEN™, CA). Zeocin-resistant transformants were selected from yeast extract peptone dextrose sorbitol medium (YPDS) agar plates containing the zeocin. PCR sequencing was used to confirm insertion of the cystatin C gene in Pichia clones.
EXAMPLE 3 Expression and Purification of Modified Human Cystatin C
The Pichia transformants were incubated in yeast minimal medium (YMM). The Pichia transformants were grown at 30° C. for one day in 5 mL of YMM, and then subcultured at 30° C. for four days in 500 mnl of fresh YMM. 100% methanol was added into the fresh YMM to a final concentration of 0.5% methanol every 24 hours to maintain induction of the cystatin gene.
Recombinant modified human cystatin C was secreted in the Pichia culture media. The extracellular proteins were collected using an ultrafiltration system with 10,000 MW cut-off (PELLICON™ cassette filter, MILLIPORE™, Bedford, Mass.). The crude proteins thus recovered were applied to a column of Q-SEPHAROSE FAST FLOW™ (PHARMACIA™, Upsala, Sweden) equilibrated with a linear gradient of 0-0.5 M NaCl in 20 mM Tris-HCI buffer (pH7.5). The fraction including the cystatin was determined by the inhibitory activity against papain as described below. The fraction was applied to a column of sephacryl S-100 HR (PHARMACIA™) equilibrated with 0.15 M NaCl-20 mM Tris-HCI buffer (pH7.5). The fraction which showed the inhibitory activity was collected.
EXAMPLE 4 Assay of Inhibitory Activity and Heat Resistance of Modified Human Cysain C
Cystatin activity was assayed by measuring papain inhibitory activity using Nα-Benzoyl-DL-Arg-p-Nitroanilide (Bz—Arg—NA) (34). 0.1 ml of cystatin sample and 0.1 ml of papain solution (0.5 mg/ml) were pipetted into 0.1 mL of 50 mM Tris-HCI buffer (pH7.5) containing 100 mM Bz—Arg—NA, 2 mM EDTA, and 5 mM cystein. The solution was incubated for 25 min at 37° C. The reaction was stopped with 0.2 ml of 30% acetic acid, and the nitroaniline liberated by enzymatic activity is quantified by measurement of light absorption of the solution at 410nm. The inhibitory activity was expressed as the amount of enzyme (mg) inhibited by 1 mg of inhibitor (U/mg).
Heat resistance assays were performed by heating a solution containing a known amount of cystatin to a controlled temperature for a controlled time; cooling the solution; mixing the cooled cystatin solution with a known amount of papain and protein substrate, and measuring turbidity of the mixture. The recombinant cystatins were heated to 95° C. at a rate of 1° C./min for 30° C. in 50 mM sodium phosphate buffer (pH 7.5). Protein concentration was 1 mg/ml.
At preset temperatures, each heated sample was transferred into a cuvette and the turbidity measured at 500 nm. The residual papain-inhibiting activity of the heated samples was also measured as described above. This procedure was repeated in triplicate.
The above examples are provided by way of illustration only and are in no way intended to limit the scope of the invention. One of skill in the art will see that the invention may be modified in various ways without departing from the spirit or principle of the invention. We claim all such modifications.
References
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9. Collins et al., (1998) Oral Microbiol Immunol. 13 (1), 59-61
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16. Atkinson et al., (1996) PCT Patent No. WO 96/116173
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33. Invitrogen Product Catalogue, 1998. Invitrogen, Carlsbad, Calif.
34. Barrett et al., (1981) Methods. Enzymol. 80: 771-778
                  
#             SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS:  32
<210> SEQ ID NO 1
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Ser Ser Pro Gly Lys Pro Pro Arg Leu Val Gl
#y Gly Pro Met Asp Ala
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#                 10 
#                 15
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Ser Val Glu Glu Glu Gly Val Arg Arg Ala Le
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             20     
#             25     
#             30
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Glu Tyr Asn Lys Ala Ser Asn Asp Met Tyr Hi
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Val Val Arg Ala Arg Lys Gln Ile Val Ala Gl
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Asp Val Glu Leu Gly Arg Thr Thr Cys Thr Ly
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# 80
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Asp Asn Cys Pro Phe His Asp Gln Pro His Le
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Cys Ser Phe Gln Ile Tyr Ala Val Pro Trp Gl
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#           105      
#           110
tcg aaa tcc acc tgt cag gac gcc tag    
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#            363
Ser Lys Ser Thr Cys Gln Asp Ala
        115          
#       120
<210> SEQ ID NO 2
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Ser Ser Pro Gly Lys Pro Pro Arg Leu Val Gl
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  1               5 
#                 10 
#                 15
Ser Val Glu Glu Glu Gly Val Arg Arg Ala Le
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             20     
#             25     
#             30
Glu Tyr Asn Lys Ala Ser Asn Asp Met Tyr Hi
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         35         
#         40         
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Val Val Arg Ala Arg Lys Gln Ile Val Ala Gl
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     50             
#     55             
#     60
Asp Val Glu Leu Gly Arg Thr Thr Cys Thr Ly
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 65                 
# 70                 
# 75                 
# 80
Asp Asn Cys Pro Phe His Asp Gln Pro His Le
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#                 90 
#                 95
Cys Ser Phe Gln Ile Tyr Ala Val Pro Trp Gl
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            100      
#           105      
#           110
Ser Lys Ser Thr Cys Gln Asp Ala
        115          
#       120
<210> SEQ ID NO 3
<211> LENGTH: 366
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<213> ORGANISM: Homo sapiens
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Ala Asp Leu Asn Asp Glu Trp Val Gln Arg Al
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             20     
#             25     
#             30
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Ser Glu Tyr Asn Lys Ala Thr Glu Asp Glu Ty
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         35         
#         40         
#         45
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Gln Val Leu Arg Ala Arg Glu Gln Thr Phe Gl
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     50             
#     55             
#     60
ttc gac gta gag gtg ggc cgc acc ata tgt ac
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Phe Asp Val Glu Val Gly Arg Thr Ile Cys Th
#r Lys Ser Gln Pro Asn
 65                 
# 70                 
# 75                 
# 80
ttg gac acc tgt gcc ttc cat gaa cag cca ga
#a ctg cag aag aaa cag      288
Leu Asp Thr Cys Ala Phe His Glu Gln Pro Gl
#u Leu Gln Lys Lys Gln
                 85 
#                 90 
#                 95
tta tgc tct ttc gag atc tac gaa gtt ccc tg
#g gag gac aga atg tcc      336
Leu Cys Ser Phe Glu Ile Tyr Glu Val Pro Tr
#p Glu Asp Arg Met Ser
            100      
#           105      
#           110
ctg gtg aat tcc agg tgt caa gaa gcc tag  
#                  
#          366
Leu Val Asn Ser Arg Cys Gln Glu Ala
        115          
#       120
<210> SEQ ID NO 4
<211> LENGTH: 121
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 4
Ser Ser Ser Lys Glu Glu Asn Arg Ile Ile Pr
#o Gly Gly Ile Tyr Asp
  1               5 
#                 10 
#                 15
Ala Asp Leu Asn Asp Glu Trp Val Gln Arg Al
#a Leu His Phe Ala Ile
             20     
#             25     
#             30
Ser Glu Tyr Asn Lys Ala Thr Glu Asp Glu Ty
#r Tyr Arg Arg Pro Leu
         35         
#         40         
#         45
Gln Val Leu Arg Ala Arg Glu Gln Thr Phe Gl
#y Gly Val Asn Tyr Phe
     50             
#     55             
#     60
Phe Asp Val Glu Val Gly Arg Thr Ile Cys Th
#r Lys Ser Gln Pro Asn
 65                 
# 70                 
# 75                 
# 80
Leu Asp Thr Cys Ala Phe His Glu Gln Pro Gl
#u Leu Gln Lys Lys Gln
                 85 
#                 90 
#                 95
Leu Cys Ser Phe Glu Ile Tyr Glu Val Pro Tr
#p Glu Asp Arg Met Ser
            100      
#           105      
#           110
Leu Val Asn Ser Arg Cys Gln Glu Ala
        115          
#       120
<210> SEQ ID NO 5
<211> LENGTH: 366
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (1)..(366)
<400> SEQUENCE: 5
tgg agc ccc aag gag gag gat agg ata atc cc
#g ggt ggc atc tat aac       48
Trp Ser Pro Lys Glu Glu Asp Arg Ile Ile Pr
#o Gly Gly Ile Tyr Asn
  1               5 
#                 10 
#                 15
gca gac ctc aat gat gag tgg gta cag cgt gc
#c ctt cac ttc gcc atc       96
Ala Asp Leu Asn Asp Glu Trp Val Gln Arg Al
#a Leu His Phe Ala Ile
             20     
#             25     
#             30
agc gag tat aac aag gcc acc aaa gat gac ta
#c tac aga cgt ccg ctg      144
Ser Glu Tyr Asn Lys Ala Thr Lys Asp Asp Ty
#r Tyr Arg Arg Pro Leu
         35         
#         40         
#         45
cgg gta cta aga gcc agg caa cag acc gtt gg
#g ggg gtg aat tac ttc      192
Arg Val Leu Arg Ala Arg Gln Gln Thr Val Gl
#y Gly Val Asn Tyr Phe
     50             
#     55             
#     60
ttc gac gta gag gtg ggc cga acc ata tgt ac
#c aag tcc cag ccc aac      240
Phe Asp Val Glu Val Gly Arg Thr Ile Cys Th
#r Lys Ser Gln Pro Asn
 65                 
# 70                 
# 75                 
# 80
ttg gac acc tgt gcc ttc cat gaa cag cca ga
#a ctg cag aag aaa cag      288
Leu Asp Thr Cys Ala Phe His Glu Gln Pro Gl
#u Leu Gln Lys Lys Gln
                 85 
#                 90 
#                 95
ttg tgc tct ttc gag atc tac gaa gtt ccc tg
#g gag aac aga agg tcc      336
Leu Cys Ser Phe Glu Ile Tyr Glu Val Pro Tr
#p Glu Asn Arg Arg Ser
            100      
#           105      
#           110
ctg gtg aaa tcc agg tgt caa gaa tcc tag  
#                  
#          366
Leu Val Lys Ser Arg Cys Gln Glu Ser
        115          
#       120
<210> SEQ ID NO 6
<211> LENGTH: 121
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 6
Trp Ser Pro Lys Glu Glu Asp Arg Ile Ile Pr
#o Gly Gly Ile Tyr Asn
  1               5 
#                 10 
#                 15
Ala Asp Leu Asn Asp Glu Trp Val Gln Arg Al
#a Leu His Phe Ala Ile
             20     
#             25     
#             30
Ser Glu Tyr Asn Lys Ala Thr Lys Asp Asp Ty
#r Tyr Arg Arg Pro Leu
         35         
#         40         
#         45
Arg Val Leu Arg Ala Arg Gln Gln Thr Val Gl
#y Gly Val Asn Tyr Phe
     50             
#     55             
#     60
Phe Asp Val Glu Val Gly Arg Thr Ile Cys Th
#r Lys Ser Gln Pro Asn
 65                 
# 70                 
# 75                 
# 80
Leu Asp Thr Cys Ala Phe His Glu Gln Pro Gl
#u Leu Gln Lys Lys Gln
                 85 
#                 90 
#                 95
Leu Cys Ser Phe Glu Ile Tyr Glu Val Pro Tr
#p Glu Asn Arg Arg Ser
            100      
#           105      
#           110
Leu Val Lys Ser Arg Cys Gln Glu Ser
        115          
#       120
<210> SEQ ID NO 7
<211> LENGTH: 366
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (1)..(366)
<400> SEQUENCE: 7
tgg agc ccc cag gag gag gac agg ata atc ga
#g ggt ggc atc tat gat       48
Trp Ser Pro Gln Glu Glu Asp Arg Ile Ile Gl
#u Gly Gly Ile Tyr Asp
  1               5 
#                 10 
#                 15
gca gac ctc aat gat gag cgg gta cag cgt gc
#c ctt cac ttt gtc atc       96
Ala Asp Leu Asn Asp Glu Arg Val Gln Arg Al
#a Leu His Phe Val Ile
             20     
#             25     
#             30
agc gag tat aac aag gcc act gaa gat gag ta
#c tac aga cgc ctg ctg      144
Ser Glu Tyr Asn Lys Ala Thr Glu Asp Glu Ty
#r Tyr Arg Arg Leu Leu
         35         
#         40         
#         45
cgg gtg cta cga gcc agg gag cag atc gtg gg
#c ggg gtg aat tac ttc      192
Arg Val Leu Arg Ala Arg Glu Gln Ile Val Gl
#y Gly Val Asn Tyr Phe
     50             
#     55             
#     60
ttc gac ata gag gtg ggc cga acc ata tgt ac
#c aag tcc cag ccc aac      240
Phe Asp Ile Glu Val Gly Arg Thr Ile Cys Th
#r Lys Ser Gln Pro Asn
 65                 
# 70                 
# 75                 
# 80
ttg gac acc tgt gcc ttc cat gaa cag cca ga
#a ctg cag aag aaa cag      288
Leu Asp Thr Cys Ala Phe His Glu Gln Pro Gl
#u Leu Gln Lys Lys Gln
                 85 
#                 90 
#                 95
ttg tgc tct ttc cag atc tac gaa gtt ccc tg
#g gag gac aga atg tcc      336
Leu Cys Ser Phe Gln Ile Tyr Glu Val Pro Tr
#p Glu Asp Arg Met Ser
            100      
#           105      
#           110
ctg gtg aat tcc agg tgt caa gaa gcc tag  
#                  
#          366
Leu Val Asn Ser Arg Cys Gln Glu Ala
        115          
#       120
<210> SEQ ID NO 8
<211> LENGTH: 121
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 8
Trp Ser Pro Gln Glu Glu Asp Arg Ile Ile Gl
#u Gly Gly Ile Tyr Asp
  1               5 
#                 10 
#                 15
Ala Asp Leu Asn Asp Glu Arg Val Gln Arg Al
#a Leu His Phe Val Ile
             20     
#             25     
#             30
Ser Glu Tyr Asn Lys Ala Thr Glu Asp Glu Ty
#r Tyr Arg Arg Leu Leu
         35         
#         40         
#         45
Arg Val Leu Arg Ala Arg Glu Gln Ile Val Gl
#y Gly Val Asn Tyr Phe
     50             
#     55             
#     60
Phe Asp Ile Glu Val Gly Arg Thr Ile Cys Th
#r Lys Ser Gln Pro Asn
 65                 
# 70                 
# 75                 
# 80
Leu Asp Thr Cys Ala Phe His Glu Gln Pro Gl
#u Leu Gln Lys Lys Gln
                 85 
#                 90 
#                 95
Leu Cys Ser Phe Gln Ile Tyr Glu Val Pro Tr
#p Glu Asp Arg Met Ser
            100      
#           105      
#           110
Leu Val Asn Ser Arg Cys Gln Glu Ala
        115          
#       120
<210> SEQ ID NO 9
<211> LENGTH: 369
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (1)..(369)
<400> SEQUENCE: 9
ggg agt gcc tcg gcc caa tct agg acc ttg gc
#a ggt ggc atc cat gcc       48
Gly Ser Ala Ser Ala Gln Ser Arg Thr Leu Al
#a Gly Gly Ile His Ala
  1               5 
#                 10 
#                 15
aca gac ctc aat gac aag agt gtg cag cgt gc
#c ctg gac ttt gcc atc       96
Thr Asp Leu Asn Asp Lys Ser Val Gln Arg Al
#a Leu Asp Phe Ala Ile
             20     
#             25     
#             30
agc gag tac aac aag gtc att aat aag gat ga
#g tac tac agc cgc cct      144
Ser Glu Tyr Asn Lys Val Ile Asn Lys Asp Gl
#u Tyr Tyr Ser Arg Pro
         35         
#         40         
#         45
ctg cag gtg atg gct gcc tac cag cag atc gt
#g ggt ggg gtg aac tac      192
Leu Gln Val Met Ala Ala Tyr Gln Gln Ile Va
#l Gly Gly Val Asn Tyr
     50             
#     55             
#     60
tac ttc aat gtg aag ttc ggt cga acc aca tg
#c acc aag tcc cag ccc      240
Tyr Phe Asn Val Lys Phe Gly Arg Thr Thr Cy
#s Thr Lys Ser Gln Pro
 65                 
# 70                 
# 75                 
# 80
aac ttg gac aac tgt ccc ttc aat gac cag cc
#a aaa ctg aaa gag gaa      288
Asn Leu Asp Asn Cys Pro Phe Asn Asp Gln Pr
#o Lys Leu Lys Glu Glu
                 85 
#                 90 
#                 95
gag ttc tgc tct ttc cag atc aat gaa gtt cc
#c tgg gag gat aaa att      336
Glu Phe Cys Ser Phe Gln Ile Asn Glu Val Pr
#o Trp Glu Asp Lys Ile
            100      
#           105      
#           110
tcc att ctg aac tac aag tgc cgg aaa gtc ta
#g                  
#        369
Ser Ile Leu Asn Tyr Lys Cys Arg Lys Val
        115          
#       120
<210> SEQ ID NO 10
<211> LENGTH: 122
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 10
Gly Ser Ala Ser Ala Gln Ser Arg Thr Leu Al
#a Gly Gly Ile His Ala
  1               5 
#                 10 
#                 15
Thr Asp Leu Asn Asp Lys Ser Val Gln Arg Al
#a Leu Asp Phe Ala Ile
             20     
#             25     
#             30
Ser Glu Tyr Asn Lys Val Ile Asn Lys Asp Gl
#u Tyr Tyr Ser Arg Pro
         35         
#         40         
#         45
Leu Gln Val Met Ala Ala Tyr Gln Gln Ile Va
#l Gly Gly Val Asn Tyr
     50             
#     55             
#     60
Tyr Phe Asn Val Lys Phe Gly Arg Thr Thr Cy
#s Thr Lys Ser Gln Pro
 65                 
# 70                 
# 75                 
# 80
Asn Leu Asp Asn Cys Pro Phe Asn Asp Gln Pr
#o Lys Leu Lys Glu Glu
                 85 
#                 90 
#                 95
Glu Phe Cys Ser Phe Gln Ile Asn Glu Val Pr
#o Trp Glu Asp Lys Ile
            100      
#           105      
#           110
Ser Ile Leu Asn Tyr Lys Cys Arg Lys Val
        115          
#       120
<210> SEQ ID NO 11
<211> LENGTH: 387
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (1)..(387)
<400> SEQUENCE: 11
ctg cca cgc gat gcc cgg gcc cgg ccg cag ga
#g cgc atg gtc gga gaa       48
Leu Pro Arg Asp Ala Arg Ala Arg Pro Gln Gl
#u Arg Met Val Gly Glu
  1               5 
#                 10 
#                 15
ctc cgg gac ctg tcg ccc gac gac ccg cag gt
#g cag aag gcg gcg cag       96
Leu Arg Asp Leu Ser Pro Asp Asp Pro Gln Va
#l Gln Lys Ala Ala Gln
             20     
#             25     
#             30
gcg gcc gtg gcc agc tac aac atg ggc agc aa
#c agc atc tac tac ttc      144
Ala Ala Val Ala Ser Tyr Asn Met Gly Ser As
#n Ser Ile Tyr Tyr Phe
         35         
#         40         
#         45
cga gac acg cac atc atc aag gcg cag agc ca
#g ctg gtg gcc ggc atc      192
Arg Asp Thr His Ile Ile Lys Ala Gln Ser Gl
#n Leu Val Ala Gly Ile
     50             
#     55             
#     60
aag tac ttc ctg acg atg gag atg ggg agc ac
#a gac tgc cgc aag acc      240
Lys Tyr Phe Leu Thr Met Glu Met Gly Ser Th
#r Asp Cys Arg Lys Thr
 65                 
# 70                 
# 75                 
# 80
agg gtc act gga gac cac gtc gac ctc acc ac
#t tgc ccc ctg gca gca      288
Arg Val Thr Gly Asp His Val Asp Leu Thr Th
#r Cys Pro Leu Ala Ala
                 85 
#                 90 
#                 95
ggg gcg cag cag gag aag ctg cgc tgt gac tt
#t gag gtc ctt gtg gtt      336
Gly Ala Gln Gln Glu Lys Leu Arg Cys Asp Ph
#e Glu Val Leu Val Val
            100      
#           105      
#           110
ccc tgg cag aac tcc tct cag ctc cta aag ca
#c aac tgt gtg cag atg      384
Pro Trp Gln Asn Ser Ser Gln Leu Leu Lys Hi
#s Asn Cys Val Gln Met
        115          
#       120          
#       125
tga                  
#                  
#                  
#            387
<210> SEQ ID NO 12
<211> LENGTH: 128
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12
Leu Pro Arg Asp Ala Arg Ala Arg Pro Gln Gl
#u Arg Met Val Gly Glu
  1               5 
#                 10 
#                 15
Leu Arg Asp Leu Ser Pro Asp Asp Pro Gln Va
#l Gln Lys Ala Ala Gln
             20     
#             25     
#             30
Ala Ala Val Ala Ser Tyr Asn Met Gly Ser As
#n Ser Ile Tyr Tyr Phe
         35         
#         40         
#         45
Arg Asp Thr His Ile Ile Lys Ala Gln Ser Gl
#n Leu Val Ala Gly Ile
     50             
#     55             
#     60
Lys Tyr Phe Leu Thr Met Glu Met Gly Ser Th
#r Asp Cys Arg Lys Thr
 65                 
# 70                 
# 75                 
# 80
Arg Val Thr Gly Asp His Val Asp Leu Thr Th
#r Cys Pro Leu Ala Ala
                 85 
#                 90 
#                 95
Gly Ala Gln Gln Glu Lys Leu Arg Cys Asp Ph
#e Glu Val Leu Val Val
            100      
#           105      
#           110
Pro Trp Gln Asn Ser Ser Gln Leu Leu Lys Hi
#s Asn Cys Val Gln Met
        115          
#       120          
#       125
<210> SEQ ID NO 13
<211> LENGTH: 366
<212> TYPE: DNA
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (1)..(366)
<400> SEQUENCE: 13
cgg ccg cag gag cgc atg gtc gga gaa ctc cg
#g gac ctg tcg ccc gac       48
Arg Pro Gln Glu Arg Met Val Gly Glu Leu Ar
#g Asp Leu Ser Pro Asp
  1               5 
#                 10 
#                 15
gac ccg cag gtg cag aag gcg gcg cag gcg gc
#c gtg gcc agc tac aac       96
Asp Pro Gln Val Gln Lys Ala Ala Gln Ala Al
#a Val Ala Ser Tyr Asn
             20     
#             25     
#             30
atg ggc agc aac agc atc tac tac ttc cga ga
#c acg cac atc atc aag      144
Met Gly Ser Asn Ser Ile Tyr Tyr Phe Arg As
#p Thr His Ile Ile Lys
         35         
#         40         
#         45
gcg cag agc cag ctg gtg gcc ggc atc aag ta
#c ttc ctg acg atg gag      192
Ala Gln Ser Gln Leu Val Ala Gly Ile Lys Ty
#r Phe Leu Thr Met Glu
     50             
#     55             
#     60
atg ggg agc aca gac tgc cgc aag acc agg gt
#c act gga gac cac gtc      240
Met Gly Ser Thr Asp Cys Arg Lys Thr Arg Va
#l Thr Gly Asp His Val
 65                 
# 70                 
# 75                 
# 80
gac ctc acc act tgc ccc ctg gca gca ggg gc
#g cag cag gag aag ctg      288
Asp Leu Thr Thr Cys Pro Leu Ala Ala Gly Al
#a Gln Gln Glu Lys Leu
                 85 
#                 90 
#                 95
cgc tgt gac ttt gag gtc ctt gtg gtt ccc tg
#g cag aac tcc tct cag      336
Arg Cys Asp Phe Glu Val Leu Val Val Pro Tr
#p Gln Asn Ser Ser Gln
            100      
#           105      
#           110
ctc cta aag cac aac tgt gtg cag atg tga  
#                  
#          366
Leu Leu Lys His Asn Cys Val Gln Met
        115          
#       120
<210> SEQ ID NO 14
<211> LENGTH: 121
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 14
Arg Pro Gln Glu Arg Met Val Gly Glu Leu Ar
#g Asp Leu Ser Pro Asp
  1               5 
#                 10 
#                 15
Asp Pro Gln Val Gln Lys Ala Ala Gln Ala Al
#a Val Ala Ser Tyr Asn
             20     
#             25     
#             30
Met Gly Ser Asn Ser Ile Tyr Tyr Phe Arg As
#p Thr His Ile Ile Lys
         35         
#         40         
#         45
Ala Gln Ser Gln Leu Val Ala Gly Ile Lys Ty
#r Phe Leu Thr Met Glu
     50             
#     55             
#     60
Met Gly Ser Thr Asp Cys Arg Lys Thr Arg Va
#l Thr Gly Asp His Val
 65                 
# 70                 
# 75                 
# 80
Asp Leu Thr Thr Cys Pro Leu Ala Ala Gly Al
#a Gln Gln Glu Lys Leu
                 85 
#                 90 
#                 95
Arg Cys Asp Phe Glu Val Leu Val Val Pro Tr
#p Gln Asn Ser Ser Gln
            100      
#           105      
#           110
Leu Leu Lys His Asn Cys Val Gln Met
        115          
#       120
<210> SEQ ID NO 15
<211> LENGTH: 351
<212> TYPE: DNA
<213> ORGANISM: Gallus sp.
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (1)..(351)
<400> SEQUENCE: 15
agc gag gac cgc tcc cgg ctc ctg ggg gct cc
#a gtg cct gta gat gag       48
Ser Glu Asp Arg Ser Arg Leu Leu Gly Ala Pr
#o Val Pro Val Asp Glu
  1               5 
#                 10 
#                 15
aac gac gag ggc ttg caa cgg gcc ctg cag tt
#c gcg atg gcc gag tac       96
Asn Asp Glu Gly Leu Gln Arg Ala Leu Gln Ph
#e Ala Met Ala Glu Tyr
             20     
#             25     
#             30
aac agg gcc agc aac gat aag tac tcc agc cg
#g gtg gtg cgg gtc atc      144
Asn Arg Ala Ser Asn Asp Lys Tyr Ser Ser Ar
#g Val Val Arg Val Ile
         35         
#         40         
#         45
agc gcc aag cgg cag ctc gtg tct gga atc aa
#g tac atc ctg cag gtt      192
Ser Ala Lys Arg Gln Leu Val Ser Gly Ile Ly
#s Tyr Ile Leu Gln Val
     50             
#     55             
#     60
gag att ggt cgc aca act tgc ccc aag tca tc
#a ggt gat ctc cag agc      240
Glu Ile Gly Arg Thr Thr Cys Pro Lys Ser Se
#r Gly Asp Leu Gln Ser
 65                 
# 70                 
# 75                 
# 80
tgc gaa ttc cac gat gag cca gag atg gct aa
#g tat acc aca tgc acc      288
Cys Glu Phe His Asp Glu Pro Glu Met Ala Ly
#s Tyr Thr Thr Cys Thr
                 85 
#                 90 
#                 95
ttt gta gtg tac agt att cct tgg cta aac ca
#a att aaa ctg ctg gaa      336
Phe Val Val Tyr Ser Ile Pro Trp Leu Asn Gl
#n Ile Lys Leu Leu Glu
            100      
#           105      
#           110
agc aag tgc cag taa           
#                  
#                  
#   351
Ser Lys Cys Gln
        115
<210> SEQ ID NO 16
<211> LENGTH: 116
<212> TYPE: PRT
<213> ORGANISM: Gallus sp.
<400> SEQUENCE: 16
Ser Glu Asp Arg Ser Arg Leu Leu Gly Ala Pr
#o Val Pro Val Asp Glu
  1               5 
#                 10 
#                 15
Asn Asp Glu Gly Leu Gln Arg Ala Leu Gln Ph
#e Ala Met Ala Glu Tyr
             20     
#             25     
#             30
Asn Arg Ala Ser Asn Asp Lys Tyr Ser Ser Ar
#g Val Val Arg Val Ile
         35         
#         40         
#         45
Ser Ala Lys Arg Gln Leu Val Ser Gly Ile Ly
#s Tyr Ile Leu Gln Val
     50             
#     55             
#     60
Glu Ile Gly Arg Thr Thr Cys Pro Lys Ser Se
#r Gly Asp Leu Gln Ser
 65                 
# 70                 
# 75                 
# 80
Cys Glu Phe His Asp Glu Pro Glu Met Ala Ly
#s Tyr Thr Thr Cys Thr
                 85 
#                 90 
#                 95
Phe Val Val Tyr Ser Ile Pro Trp Leu Asn Gl
#n Ile Lys Leu Leu Glu
            100      
#           105      
#           110
Ser Lys Cys Gln
        115
<210> SEQ ID NO 17
<211> LENGTH: 336
<212> TYPE: DNA
<213> ORGANISM: Cyprinus carpio
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (1)..(336)
<400> SEQUENCE: 17
act ggg att cct gga ggc ctt gta gat gca ga
#c att aac gat aaa gat       48
Thr Gly Ile Pro Gly Gly Leu Val Asp Ala As
#p Ile Asn Asp Lys Asp
  1               5 
#                 10 
#                 15
gtt cag aag gcg tta cgc ttc gca gtg gac ca
#t tac aac ggc caa agc       96
Val Gln Lys Ala Leu Arg Phe Ala Val Asp Hi
#s Tyr Asn Gly Gln Ser
             20     
#             25     
#             30
aac gat gcg ttt gtg cgt aaa gtt tcc aaa gt
#a atc aag gtt caa caa      144
Asn Asp Ala Phe Val Arg Lys Val Ser Lys Va
#l Ile Lys Val Gln Gln
         35         
#         40         
#         45
caa gtt gcc gct ggc atg aaa tac atc ttc ac
#t gtg aag atg gaa gta      192
Gln Val Ala Ala Gly Met Lys Tyr Ile Phe Th
#r Val Lys Met Glu Val
     50             
#     55             
#     60
gcc tcc tgc aaa aag ggt gga gtt aag acc at
#g tgt gcc gtt ccg aag      240
Ala Ser Cys Lys Lys Gly Gly Val Lys Thr Me
#t Cys Ala Val Pro Lys
 65                 
# 70                 
# 75                 
# 80
aat ccc agt att gaa cag gtc att cag tgc aa
#a ata acg gtc tgg agc      288
Asn Pro Ser Ile Glu Gln Val Ile Gln Cys Ly
#s Ile Thr Val Trp Ser
                 85 
#                 90 
#                 95
cag cca tgg tta aac tcc ttg aaa gtc act ga
#a aac acc tgc atg tag      336
Gln Pro Trp Leu Asn Ser Leu Lys Val Thr Gl
#u Asn Thr Cys Met
            100      
#           105      
#           110
<210> SEQ ID NO 18
<211> LENGTH: 111
<212> TYPE: PRT
<213> ORGANISM: Cyprinus carpio
<400> SEQUENCE: 18
Thr Gly Ile Pro Gly Gly Leu Val Asp Ala As
#p Ile Asn Asp Lys Asp
  1               5 
#                 10 
#                 15
Val Gln Lys Ala Leu Arg Phe Ala Val Asp Hi
#s Tyr Asn Gly Gln Ser
             20     
#             25     
#             30
Asn Asp Ala Phe Val Arg Lys Val Ser Lys Va
#l Ile Lys Val Gln Gln
         35         
#         40         
#         45
Gln Val Ala Ala Gly Met Lys Tyr Ile Phe Th
#r Val Lys Met Glu Val
     50             
#     55             
#     60
Ala Ser Cys Lys Lys Gly Gly Val Lys Thr Me
#t Cys Ala Val Pro Lys
 65                 
# 70                 
# 75                 
# 80
Asn Pro Ser Ile Glu Gln Val Ile Gln Cys Ly
#s Ile Thr Val Trp Ser
                 85 
#                 90 
#                 95
Gln Pro Trp Leu Asn Ser Leu Lys Val Thr Gl
#u Asn Thr Cys Met
            100      
#           105      
#           110
<210> SEQ ID NO 19
<211> LENGTH: 336
<212> TYPE: DNA
<213> ORGANISM: Oncorhynchus keta
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (1)..(336)
<400> SEQUENCE: 19
ggt ttg gtc gga ggc ccc atg gac gca aat at
#g aac gac caa gga acg       48
Gly Leu Val Gly Gly Pro Met Asp Ala Asn Me
#t Asn Asp Gln Gly Thr
  1               5 
#                 10 
#                 15
aga gac gcc ctg cag ttc gcg gtg gtc gaa ca
#c aac aag aaa aca aac       96
Arg Asp Ala Leu Gln Phe Ala Val Val Glu Hi
#s Asn Lys Lys Thr Asn
             20     
#             25     
#             30
gac atg ttt gtc agg cag gtg gcc aag gtt gt
#c aat gca cag aaa cag      144
Asp Met Phe Val Arg Gln Val Ala Lys Val Va
#l Asn Ala Gln Lys Gln
         35         
#         40         
#         45
gtg gta tct ggg atg aag tac atc ttc aca gt
#g cag atg ggc agg acc      192
Val Val Ser Gly Met Lys Tyr Ile Phe Thr Va
#l Gln Met Gly Arg Thr
     50             
#     55             
#     60
cca tgc agg aag gga ggt gtt gag aag atc tg
#c tcc gtg cac aaa gac      240
Pro Cys Arg Lys Gly Gly Val Glu Lys Ile Cy
#s Ser Val His Lys Asp
 65                 
# 70                 
# 75                 
# 80
ccg cag atg gct gtg ccc tac aag tgc acc tt
#c gag gtg tgg agc cgc      288
Pro Gln Met Ala Val Pro Tyr Lys Cys Thr Ph
#e Glu Val Trp Ser Arg
                 85 
#                 90 
#                 95
ccc tgg atg agc gat atc cag atg gtc aag aa
#c cag tgt gaa agt taa      336
Pro Trp Met Ser Asp Ile Gln Met Val Lys As
#n Gln Cys Glu Ser
            100      
#           105      
#           110
<210> SEQ ID NO 20
<211> LENGTH: 111
<212> TYPE: PRT
<213> ORGANISM: Oncorhynchus keta
<400> SEQUENCE: 20
Gly Leu Val Gly Gly Pro Met Asp Ala Asn Me
#t Asn Asp Gln Gly Thr
  1               5 
#                 10 
#                 15
Arg Asp Ala Leu Gln Phe Ala Val Val Glu Hi
#s Asn Lys Lys Thr Asn
             20     
#             25     
#             30
Asp Met Phe Val Arg Gln Val Ala Lys Val Va
#l Asn Ala Gln Lys Gln
         35         
#         40         
#         45
Val Val Ser Gly Met Lys Tyr Ile Phe Thr Va
#l Gln Met Gly Arg Thr
     50             
#     55             
#     60
Pro Cys Arg Lys Gly Gly Val Glu Lys Ile Cy
#s Ser Val His Lys Asp
 65                 
# 70                 
# 75                 
# 80
Pro Gln Met Ala Val Pro Tyr Lys Cys Thr Ph
#e Glu Val Trp Ser Arg
                 85 
#                 90 
#                 95
Pro Trp Met Ser Asp Ile Gln Met Val Lys As
#n Gln Cys Glu Ser
            100      
#           105      
#           110
<210> SEQ ID NO 21
<211> LENGTH: 336
<212> TYPE: DNA
<213> ORGANISM: Oncorhynchus mykiss
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (1)..(336)
<400> SEQUENCE: 21
ggt ttg atc gga ggc ccc atg gac gca aat at
#g aac gac caa gga acg       48
Gly Leu Ile Gly Gly Pro Met Asp Ala Asn Me
#t Asn Asp Gln Gly Thr
  1               5 
#                 10 
#                 15
aga gac gcc ctg cag ttc gcg gtg gtc gaa ca
#c aac aag aaa aca aac       96
Arg Asp Ala Leu Gln Phe Ala Val Val Glu Hi
#s Asn Lys Lys Thr Asn
             20     
#             25     
#             30
gac atg ttt gtc agg cag gtg gcc aag gtt gt
#c aat gca cag aag cag      144
Asp Met Phe Val Arg Gln Val Ala Lys Val Va
#l Asn Ala Gln Lys Gln
         35         
#         40         
#         45
gtg gta tct ggg atg aag tac atc ttc aca gt
#g cag atg ggc agg acc      192
Val Val Ser Gly Met Lys Tyr Ile Phe Thr Va
#l Gln Met Gly Arg Thr
     50             
#     55             
#     60
cca tgc agg aag gga ggt gtt gag aag gtc tg
#c tcc gtg cac aag gac      240
Pro Cys Arg Lys Gly Gly Val Glu Lys Val Cy
#s Ser Val His Lys Asp
 65                 
# 70                 
# 75                 
# 80
cca cag atg gct gtg ccc tac aag tgc acc tt
#c gag gtg tgg agc cgc      288
Pro Gln Met Ala Val Pro Tyr Lys Cys Thr Ph
#e Glu Val Trp Ser Arg
                 85 
#                 90 
#                 95
ccc tgg atg agc gat atc cag atg gtc aag aa
#c cag tgt gaa agt taa      336
Pro Trp Met Ser Asp Ile Gln Met Val Lys As
#n Gln Cys Glu Ser
            100      
#           105      
#           110
<210> SEQ ID NO 22
<211> LENGTH: 111
<212> TYPE: PRT
<213> ORGANISM: Oncorhynchus mykiss
<400> SEQUENCE: 22
Gly Leu Ile Gly Gly Pro Met Asp Ala Asn Me
#t Asn Asp Gln Gly Thr
  1               5 
#                 10 
#                 15
Arg Asp Ala Leu Gln Phe Ala Val Val Glu Hi
#s Asn Lys Lys Thr Asn
             20     
#             25     
#             30
Asp Met Phe Val Arg Gln Val Ala Lys Val Va
#l Asn Ala Gln Lys Gln
         35         
#         40         
#         45
Val Val Ser Gly Met Lys Tyr Ile Phe Thr Va
#l Gln Met Gly Arg Thr
     50             
#     55             
#     60
Pro Cys Arg Lys Gly Gly Val Glu Lys Val Cy
#s Ser Val His Lys Asp
 65                 
# 70                 
# 75                 
# 80
Pro Gln Met Ala Val Pro Tyr Lys Cys Thr Ph
#e Glu Val Trp Ser Arg
                 85 
#                 90 
#                 95
Pro Trp Met Ser Asp Ile Gln Met Val Lys As
#n Gln Cys Glu Ser
            100      
#           105      
#           110
<210> SEQ ID NO 23
<211> LENGTH: 357
<212> TYPE: DNA
<213> ORGANISM: Bos taurus
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (1)..(357)
<400> SEQUENCE: 23
cag ggc cct agg aag ggt cgc ctg ctg ggc gg
#c ctg atg gag gcg gac       48
Gln Gly Pro Arg Lys Gly Arg Leu Leu Gly Gl
#y Leu Met Glu Ala Asp
  1               5 
#                 10 
#                 15
gtc aat gag gag ggc gtg cag gag gcg ctg tc
#c ttt gcg gtc agc gag       96
Val Asn Glu Glu Gly Val Gln Glu Ala Leu Se
#r Phe Ala Val Ser Glu
             20     
#             25     
#             30
ttc aac aag cgg agc aac gac gct tac cag ag
#c cgc gtg gtg cgc gtg      144
Phe Asn Lys Arg Ser Asn Asp Ala Tyr Gln Se
#r Arg Val Val Arg Val
         35         
#         40         
#         45
gtg cgc gcc cgc aag cag gtc gtg tca ggg at
#g aac tat ttc ttg gac      192
Val Arg Ala Arg Lys Gln Val Val Ser Gly Me
#t Asn Tyr Phe Leu Asp
     50             
#     55             
#     60
gtg gag ctt ggc cgg act aca tgt acc aag tc
#c cag gcc aac ttt gac      240
Val Glu Leu Gly Arg Thr Thr Cys Thr Lys Se
#r Gln Ala Asn Phe Asp
 65                 
# 70                 
# 75                 
# 80
agc tgt ccc ttc cat aac cag ccg cac ctg aa
#g agg gaa aag ctg tgc      288
Ser Cys Pro Phe His Asn Gln Pro His Leu Ly
#s Arg Glu Lys Leu Cys
                 85 
#                 90 
#                 95
tcc ttc cag gtt tac gtc gtc cca tgg atg aa
#c acc atc aac ctg gtg      336
Ser Phe Gln Val Tyr Val Val Pro Trp Met As
#n Thr Ile Asn Leu Val
            100      
#           105      
#           110
aag ttt agc tgc cag gat taa       
#                  
#                 357
Lys Phe Ser Cys Gln Asp
        115
<210> SEQ ID NO 24
<211> LENGTH: 118
<212> TYPE: PRT
<213> ORGANISM: Bos taurus
<400> SEQUENCE: 24
Gln Gly Pro Arg Lys Gly Arg Leu Leu Gly Gl
#y Leu Met Glu Ala Asp
  1               5 
#                 10 
#                 15
Val Asn Glu Glu Gly Val Gln Glu Ala Leu Se
#r Phe Ala Val Ser Glu
             20     
#             25     
#             30
Phe Asn Lys Arg Ser Asn Asp Ala Tyr Gln Se
#r Arg Val Val Arg Val
         35         
#         40         
#         45
Val Arg Ala Arg Lys Gln Val Val Ser Gly Me
#t Asn Tyr Phe Leu Asp
     50             
#     55             
#     60
Val Glu Leu Gly Arg Thr Thr Cys Thr Lys Se
#r Gln Ala Asn Phe Asp
 65                 
# 70                 
# 75                 
# 80
Ser Cys Pro Phe His Asn Gln Pro His Leu Ly
#s Arg Glu Lys Leu Cys
                 85 
#                 90 
#                 95
Ser Phe Gln Val Tyr Val Val Pro Trp Met As
#n Thr Ile Asn Leu Val
            100      
#           105      
#           110
Lys Phe Ser Cys Gln Asp
        115
<210> SEQ ID NO 25
<211> LENGTH: 115
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial 
#Sequence: first of
      four oligonucleotides used to create
# a nucleotide
      coding for modified human cystatin 
#C
<400> SEQUENCE: 25
gtatctctcg agaaaagatc ttctccaggt aagccaccaa gattggtcgg tg
#gtccaatg     60
gacgcctctg tcgaggagga gggtgtcaga agagccttgg acttcgccgt cg
#gtg         115
<210> SEQ ID NO 26
<211> LENGTH: 115
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial 
#Sequence: the second
      of four oligonucleotides used to 
#create a nucleotide
      coding for synthetic human cystatin 
#C
<400> SEQUENCE: 26
caagaagtag ttgacaccgg cgacaatttg ctttctggct ctgacgactt gc
#aaggctct     60
ggagtggtac atgtcgttag aggccttgtt gtactcaccg acggcgaagt cc
#aag         115
<210> SEQ ID NO 27
<211> LENGTH: 115
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial 
#Sequence: the third
      of four oligonucleotides used to 
#create a nucleotide
      coding for synthetic human cystatin 
#C
<400> SEQUENCE: 27
caaattgtcg ccggtgtcaa ctacttcttg gacgttgagt tgggtagaac ta
#cttgtact     60
aagactcaac caaacttgac taactgtcca ttccacgacc aaccacactt ga
#aga         115
<210> SEQ ID NO 28
<211> LENGTH: 115
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial 
#Sequence: the fourth
      of four oligonucleotides used to 
#create a nucleotide
      coding for synthetic human cystatin 
#C
<400> SEQUENCE: 28
tgttctagat caggcgtctt gacaagtaga cttagacaaa gtcatagtac ct
#tgccatgg     60
gacggcgtaa atttggaaag aacagaaggc ctttctcttc aagtgtggtt gg
#tcg         115
<210> SEQ ID NO 29
<211> LENGTH: 30
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial 
#Sequence: forward
      primer used in site-directed mutagen
#esis to intro. a
      glycosylation site at residue 35 
#of a modified human cystatin C
<400> SEQUENCE: 29
ggtgagtaca acaagtcctc taacgacatg         
#                  
#           30
<210> SEQ ID NO 30
<211> LENGTH: 30
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial 
#Sequence: reverse
      primer used in site-directed mutagen
#esis to intro. a
      glycosylation site at residue 35 
#of a modified human cystatin C
<400> SEQUENCE: 30
catgtcgtta gaggacttgt tgtactcacc         
#                  
#           30
<210> SEQ ID NO 31
<211> LENGTH: 30
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial 
#Sequence: forward
      primer used in site-directed mutagen
#esis to intro. a
      glycosylation site at residue 36 
#of a modified human cystatin C
<400> SEQUENCE: 31
ggtgagtaca acaacgcctc taacgacatg         
#                  
#           30
<210> SEQ ID NO 32
<211> LENGTH: 30
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial 
#Sequence: reverse
      primer used in site-directed mutagen
#esis to intro. a
      glycosylation site at residue 36 
#of a modified human cystatin C
<400> SEQUENCE: 32
catgtcgtta gaggcgttgt tgtactcacc         
#                  
#           30

Claims (4)

What is claimed:
1. A modified human cystatin C comprising at least one modification of native human cystatin C (SEQ ID NO:2) selected from the group consisting of: Lys (36) Asn, Ala (37) Ser, Ala (37) Thr, Asp (81) Ser, and Asp (81) Thr.
2. The modified human cystatin C of claim 1, wherein said at least one modification increases the heat stability of the modified human cystatin C.
3. The modified human cystatin C of claim 1, wherein said at least one modification is Ala (37) Ser or Ala (37) Thr, and Asp (81) Ser or Asp (81) Thr.
4. The modified human cystatin C of claim 1, said at least one modification is Ala (37) Ser or Asp (81) Thr.
US09/775,932 1998-08-05 2001-02-02 Production and use of modified cystatins Expired - Fee Related US6534477B2 (en)

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US09/775,932 US6534477B2 (en) 1998-08-05 2001-02-02 Production and use of modified cystatins

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GB0009124D0 (en) * 2000-04-14 2000-05-31 Univ Belfast Agent against periodontal disease
ATE406174T1 (en) 2000-04-14 2008-09-15 Philip-John Lamey TREATMENT OF MIGRAINES AND VASODILATION
CN104039962B (en) * 2012-01-09 2016-06-22 苏州工业园区为真生物医药科技有限公司 The mark of breast cancer diagnosis and indication

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Cited By (8)

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Publication number Priority date Publication date Assignee Title
US20050267021A1 (en) * 2003-10-15 2005-12-01 Schiemann William P Cystatin C as an antagonist of TGF-beta and methods related thereto
WO2005037221A3 (en) * 2003-10-15 2007-05-10 Nat Jewish Med & Res Center Cystatin c as an antagonist of tgf-b and methods related thereto
US7282477B2 (en) * 2003-10-15 2007-10-16 National Jewish Medical And Research Center Cystatin C as an antagonist of TGF-β and methods related thereto
US20090093401A1 (en) * 2003-10-15 2009-04-09 National Jewish Medical And Research Center Cystatin c as an antagonist of tgf-beta and methods related thereto
US7749958B2 (en) 2003-10-15 2010-07-06 National Jewish Health Cystatin C as an antagonist of TGF-β and methods related thereto
US20100267644A1 (en) * 2003-10-15 2010-10-21 National Jewish Health Cystatin C as an Antagonist of TGF-BETA and Methods Related Thereto
US8058396B2 (en) 2003-10-15 2011-11-15 National Jewish Health Cystatin C as an antagonist of TGF-β and methods related thereto
AU2004281152B2 (en) * 2003-10-15 2012-01-19 National Jewish Health Cystatin C as an antagonist of tgf-b and methods related thereto

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WO2000008159A3 (en) 2000-05-11
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WO2000008159A2 (en) 2000-02-17
CA2335344A1 (en) 2000-02-17
EP1123399A2 (en) 2001-08-16

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